Production of gamma linolenic acid by a delta6-desaturase

ABSTRACT

Linoleic acid is converted into γ-linolenic acid by the enzyme Δ 6 -desaturase. The present invention is directed to isolated nucleic acids comprising the Δ 6 -desaturase gene. More particularly, the isolated nucleic acid comprises the promoter, coding region and termination regions of the Δ 6 -desaturase gene. The present invention provides recombinant constructions comprising the Δ 6 -desaturase coding region in functional combination with heterologous regulatory sequences. The nucleic acids and recombinant constructions of the instant invention are useful in the production of GLA in transgenic organisms.

[0001] This is a continuation-in-part of U.S. Ser. No. 08/789,936 filedJan. 28, 1997 which is a continuation-in-part of U.S. Ser. No.08/307,382,filed Sep. 14, 1994 which is a continuation of U.S. Ser. No.07/959,952 filed Oct. 13, 1992 which is a continuation-in-part of U.S.Ser. No. 817,919, filed Jan. 8, 1992, which is a continuation-in-partapplication of U.S. Ser. No. 774,475 filed Oct. 10, 1991.

FIELD OF THE INVENTION

[0002] Linoleic acid (18:2) (LA) is transformed into gamma linolenicacid (18:3) (GLA) by the enzyme Δ6-desaturase. When this enzyme, or thenucleic acid encoding it, is transferred into LA-producing cells, GLA isproduced. The present invention provides nucleic acids comprising theΔ6-desaturase gene. More specifically, the nucleic acids comprise thepromoters, coding regions and termination regions of the Δ6-desaturasegenes. The present invention is further directed to recombinantconstructions comprising a Δ6-desaturase coding region in functionalcombination with heterologous regulatory sequences. The nucleic acidsand recombinant constructions of the instant invention are useful in theproduction of GLA in transgenic organisms.

BACKGROUND OF THE INVENTION

[0003] Unsaturated fatty acids such as linoleic (C₁₈Δ^(9, 12)) andα-linolenic (C₁₈Δ^(9, 12, 15)) acids are essential dietary constituentsthat cannot be synthesized by vertebrates since vertebrate cells canintroduce double bonds at the Δ⁹ position of fatty acids but cannotintroduce additional double bonds between the Δ⁹ double bond and themethyl-terminus of the fatty acid chain. Because they are precursors ofother products, linoleic and a-linolenic acids are essential fattyacids, and are usually obtained from plant sources. Linoleic acid can beconverted by mammals into γ-linolenic acid (GLA, C₁₈Δ^(6, 9, 12) ) whichcan in turn be converted to arachidonic acid (20:4), a criticallyimportant fatty acid since it is an essential precursor of mostprostaglandins.

[0004] The dietary provision of linoleic acid, by virtue of itsresulting conversion to GLA and arachidonic acid, satisfies the dietaryneed for GLA and arachidonic acid. However, a relationship has beendemonstrated between consumption of saturated fats and health risks suchas hypercholesterolemia, atherosclerosis and other clinical disorderswhich correlate with susceptibility to coronary disease, while theconsumption of unsaturated fats has been associated with decreased bloodcholesterol concentration and reduced risk of atherosclerosis. Thetherapeutic benefits of dietary GLA may result from GLA being aprecursor to arachidonic acid and thus subsequently contributing toprostaglandin synthesis. Accordingly, consumption of the moreunsaturated GLA, rather than linoleic acid, has potential healthbenefits. However, GLA is not present in virtually any commerciallygrown crop plant.

[0005] Linoleic acid is converted into GLA by the enzyme Δ6-desaturase.Δ6-desaturase, an enzyme of more than 350 amino acids, has amembrane-bound domain and an active site for desaturation of fattyacids. When this enzyme is transferred into cells which endogenouslyproduce linoleic acid but not GLA, GLA is produced. The presentinvention, by providing genes encoding Δ6-desaturase, allows theproduction of transgenic organisms which contain functionalΔ6-desaturase and which produce GLA. In addition to allowing productionof large amounts of GLA, the present invention provides new dietarysources of GLA.

SUMMARY OF THE INVENTION

[0006] The present invention is directed to isolated Δ6-desaturasegenes. Specifically, the isolated genes comprise the Δ6-desaturasepromoters, coding regions, and termination regions.

[0007] The present invention is further directed to expression vectorscomprising the Δ6-desaturase promoter, coding region and terminationregion.

[0008] Yet another aspect of this invention is directed to expressionvectors comprising a Δ6-desaturase coding region in functionalcombination with heterologous regulatory regions, i.e. elements notderived from the Δ6-desaturase gene.

[0009] Cells and organisms comprising the vectors of the presentinvention, and progeny of such organisms, are also provided by thepresent invention.

[0010] A further aspect of the present invention provides isolatedbacterial Δ6-desaturase. Isolated plant Δ6-desaturases are alsoprovided.

[0011] Yet another aspect of this invention provides a method forproducing plants with increased gamma linolenic acid content.

[0012] A method for producing chilling tolerant plants is also providedby the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0013]FIG. 1 depicts the hydropathy profiles of the deduced amino acidsequences of Synechocystis Δ6-desaturase (Panel A) and Δ12-desaturase(Panel B). Putative membrane spanning regions are indicated by solidbars. Hydrophobic index was calculated for a window size of 19 aminoacid residues [Kyte, et al. (1982) J. Molec. Biol. 157].

[0014]FIG. 2 provides gas liquid chromatography profiles of wild type(Panel A) and transgenic (Panel B) Anabaena.

[0015]FIG. 3 is a diagram of maps of cosmid cSy75, cSy13 and Csy7 withoverlapping regions and subclones. The origins of subclones of Csy75,Csy75-3.5 and Csy7 are indicated by the dashed diagonal lines.Restriction sites that have been inactivated are in parentheses.

[0016]FIG. 4 provides gas liquid chromatography profiles of wild type(Panel A) and transgenic (Panel B) tobacco.

[0017]FIG. 5A depicts the DNA sequence of a Δ6-desaturase cDNA isolatedfrom borage.

[0018]FIG. 5B depicts the protein sequence of the open reading frame inthe isolated borage Δ6-desaturase cDNA. Three amino acid motifscharacteristic of desaturases are indicated and are, in order, lipidbox, metal box 1, and metal box 2.

[0019]FIG. 6 is a dendrogram showing similarity of the borageΔ6-desaturase to other membrane-bound desaturases. The amino acidsequence of the borage Δ6-desaturase was compared to other knowndesaturases using Gene Works (IntelliGenetics). Numerical valuescorrelate to relative phylogenetic distances between subgroups compared.

[0020]FIG. 7 is a restriction map of 221.Δ6.NOS and 121.Δ6.NOS. In221.Δ6.NOS, the remaining portion of the plasmid is pBI221 and in121.Δ6.NOS, the remaining portion of the plasmid is pBI121.

[0021]FIG. 8 provides gas liquid chromatography profiles of mocktransfected (Panel A) and 221.Δ6.NOS transfected (Panel B) carrot cells.The positions of 18:2, 18:3 a, and 18:3 γ(GLA) are indicated.

[0022]FIG. 9 provides gas liquid chromatography profiles of anuntransformed tobacco leaf (Panel A) and a tobacco leaf transformed with121.Δ6.NOS. The positions of 18:2, 18:3 a, 18:3γ (GLA), and 18:4 areindicated.

[0023]FIG. 10 is the complete DNA sequence and deduced amino acidsequence of evening primrose Δ6-desaturase. A heme binding motif ofcytochrome b5 proteins is indicated by underlined bold text. Underlinedplain text indicates three histine rich motifs (HRMs). The motifs inthis sequence are identical to those found in borage Δ6-desaturase withthe exception of those that are italicized (S 161 and L 374).

[0024]FIG. 11 is a formatted alignment of the evening primrose andborage Δ6-desaturase amino acid sequences.

[0025]FIG. 12A is a Kyte-Doolittle hydrophobicity plot for borageΔ6-desaturase.

[0026]FIG. 12B is a Kyte-Doolittle hydrophobicity plot for eveningprimrose Δ6-desaturase.

[0027]FIG. 13A is a Hopwood hydrophobicity plot for borageΔ6-desaturase. The y axis is a normalized parameter that estimateshydrophobicity; that the x axis represents the linear amino acidsequences.

[0028]FIG. 13B is a Hopwood hydrophobicity plot for evening primroseΔ6-desaturase. X and y axes are as in FIG. 13A.

[0029]FIG. 14A graphically depicts the location of the transmembraneregions for borage Δ6-desaturase. Positive values (y-axis) greater than500 are considered significant predictors of a membrane spanning region.The x-axis represents the linear amino acid sequences.

[0030]FIG. 14B graphically depicts the location of the transmembraneregions for evening primrose Δ6-desaturase. X and y axes are as in FIG.14A.

DETAILED DESCRIPTION OF THE INVENTION

[0031] The present invention provides isolated nucleic acids encodingΔ6-desaturase. To identify a nucleic acid encoding Δ6-desaturase, DNA isisolated from an organism which produces GLA. Said organism can be, forexample, an animal cell, certain fungi (e.g. Mortierella), certainbacteria (e.g. Synechocystis) or certain plants (borage, Oenothera,currants). The isolation of genomic DNA can be accomplished by a varietyof methods well-known to one of ordinary skill in the art, asexemplified by Sambrook et al. (1989) in Molecular Cloning: A LaboratoryManual, Cold Spring Harbor, NY. The isolated DNA is fragmented byphysical methods or enzymatic digestion and cloned into an appropriatevector, e.g. a bacteriophage or cosmid vector, by any of a variety ofwell-known methods which can be found in references such as Sambrook etal. (1989). Expression vectors containing the DNA of the presentinvention are specifically contemplated herein. DNA encodingA-desaturase can be identified by gain of function analysis. The vectorcontaining fragmented DNA is transferred, for example by infection,transconjugation, transfection, into a host organism that produceslinoleic acid but not GLA. As used herein, “transformation” refersgenerally to the incorporation of foreign DNA into a host cell. Methodsfor introducing recombinant DNA into a host organism are known to one ofordinary skill in the art and can be found, for example, in Sambrook etal. (1989). Production of GLA by these organisms (i.e., gain offunction) is assayed, for example by gas chromatography or other methodsknown to the ordinarily skilled artisan. Organisms which are induced toproduce GLA, i.e. have gained function by the introduction of thevector, are identified as expressing DNA encoding Δ-desaturase, and saidDNA is recovered from the organisms. The recovered DNA can again befragmented, cloned with expression vectors, and functionally assessed bythe above procedures to define with more particularity the DNA encodingΔ6-desaturase.

[0032] As an example of the present invention, random DNA is isolatedfrom the cyanobacteria Synechocystis Pasteur Culture Collection (PCC)6803, American Type Culture Collection (ATCC) 27184, cloned into acosmid vector, and introduced by transconjugation into the GLA-deficientCyanobacterium Anabaena strain PCC 7120, ATCC 27893. Production of GLAfrom Anabaena linoleic acid is monitored by gas chromatography and thecorresponding DNA fragment is isolated.

[0033] The isolated DNA is sequenced by methods well-known to one ofordinary skill in the art as found, for example, in Sambrook et al.(1989).

[0034] In accordance with the present invention, DNA moleculescomprising Δ6-desaturase genes have been isolated. More particularly, a3.588 kilobase (kb) DNA comprising a Δ6-desaturase gene has beenisolated from the cyanobacteria Synechocystis. The nucleotide sequenceof the 3.588 kb DNA was determined and is shown in SEQ ID NO:1. Openreading frames defining potential coding regions are present fromnucleotide 317 to 1507 and from nucleotide 2002 to 3081. To define thenucleotides responsible for encoding Δ6-desaturase, the 3.588 kbfragment that confers Δ6-desaturase activity is cleaved into twosubfragments, each of which contains only one open reading frame.Fragment ORF1 contains nucleotides 1 through 1704, while fragment ORF2contains nucleotides 1705 through 3588. Each fragment is subcloned inboth forward and reverse orientations into a conjugal expression vector(AM542, Wolk et al. [1984] Proc. Natl. Acad. Sci. USA 81, 1561) thatcontains a cyanobacterial carboxylase promoter. The resulting constructs(i.e. ORF1(F), ORF1(R), ORF2(F) and ORF2(R)] are conjugated to wild-typeAnabaena PCC 7120 by standard methods (see, for example, Wolk et al.(1984) Proc. Natl. Acad. Sci. USA 81, 1561). Conjugated cells ofAnabaena are identified as Neo^(R) green colonies on a brown backgroundof dying non-conjugated cells after two weeks of growth on selectivemedia (standard mineral media BG11N+containing 30 μg/ml of neomycinaccording to Rippka et al., (1979) J. Gen Microbiol. 111, 1). The greencolonies are selected and grown in selective liquid media (BG11N+with 15μg/ml neomycin). Lipids are extracted by standard methods (e.g. Dahmeret al., (1989) Journal of American Oil Chemical Society 66, 543) fromthe resulting transconjugants containing the forward and reverseoriented ORF1 and ORF2 constructs. For comparison, lipids are alsoextracted from wild-type cultures of Anabaena and Synechocystis. Thefatty acid methyl esters are analyzed by gas liquid chromatography(GLC), for example with a Tracor-560 gas liquid chromatograph equippedwith a hydrogen flame ionization detector and a capillary column. Theresults of GLC analysis are shown in Table 1. TABLE 1 Occurrence of C18fatty acids in wild-type and tranegenic cyanobacteria SOURCE 18:0 18:118:2 18:3 18:3 18:4 Anabaena + + + − + − (wild type) Anabaena + ORF1(F) + + + − + − Anabaena + ORF1 (R) + + + − + − Anabaena + ORF2(F) + + + + + + Anabaena + ORF2 (R) + + + − + − Synechocystis + + + + −− (wild type)

[0035] As assessed by GLC analysis, GLA deficient Anabaena gain thefunction of GLA production when the construct containing ORF2 in forwardorientation is introduced by transconjugation. Transconjugantscontaining constructs with ORF2 in reverse orientation to thecarboxylase promoter, or ORF1 in either orientation, show no GLAproduction. This analysis demonstrates that the single open readingframe (ORF2) within the 1884 bp fragment encodes Δ6-desaturase. The 1884bp fragment is shown as SEQ ID NO:3. This is substantiated by theoverall similarity of the hydropathy profiles between Δ6-desaturase andΔ12-desaturase [Wada et al. (1990) Nature 347] as shown in FIG. 1 as (A)and (B), respectively.

[0036] Also in accordance with the present invention, a cDNA comprisinga Δ6-desaturase gene from borage (Borago officinalis) has been isolated.The nucleotide sequence of the 1.685 kilobase (kb) cDNA was determinedand is shown in FIG. 5A (SEQ ID NO: 4). The ATG start codon and stopcodon are underlined. The amino acid sequence corresponding to the openreading frame in the borage delta 6-desaturase is shown in FIG. 5B (SEQID NO: 5).

[0037] Additionally, the present invention provides a Δ6-desaturase genefrom evening primrose (Oenothera biennis). The nucleotide sequence ofthe 1.687 kb cDNA was determined and is depicted in FIG. 10 (SEQ IDNO:26). Also shown in FIG. 10 is the deduced amino acid sequence ofevening primrose Δ6-desaturase.

[0038] Isolated nucleic acids encoding Δ6-desaturase can be identifiedfrom other GLA-producing organisms by the gain of function analysisdescribed above, or by nucleic acid hybridization techniques using theisolated nucleic acid which encodes Synechocystis, borage, or eveningprimrose Δ6-desaturase as a hybridization probe. Both methods are knownto the skilled artisan and are contemplated by the present invention.The hybridization probe can comprise the entire DNA sequence disclosedas SEQ. ID NO:1 or SEQ. ID NO:4, or a restriction fragment or other DNAfragment thereof, including an oligonucleotide probe. Methods forcloning homologous genes by cross-hybridization are known to theordinarily skilled artisan and can be found, for example, in Sambrook(1989) and Beltz et al . (1983) Methods in Enzymology 100, 266.

[0039] In another method of identifying a delta 6-desaturase gene froman organism producing GLA, a cDNA library is made from poly-A⁺RNAisolated from polysomal RNA. In order to eliminate hyper-abundantexpressed genes from the cDNA population, cDNAs or fragments thereofcorresponding to hyper-abundant cDNAs genes are used as hybridizationprobes to the cDNA library. Non hybridizing plaques are excised and theresulting bacterial colonies are used to inoculate liquid cultures andsequenced. For example, as a means of eliminating other seed storageprotein cDNAs from a cDNA library made from borage polysomal RNA, cDNAscorresponding to abundantly expressed seed storage proteins are firsthybridized to the cDNA library. The “subtracted” DNA library is thenused to generate expressed sequence tags (ETSs) and such tags are usedto scan a data base such as GenBank to identify potential desaturates.

[0040] Using another method, an evening primrose CDNA may be isolated byfirst synthesizing sequences from the borage Δ6-desaturase gene and thenusing these sequences as primers in a PCR reaction with the eveningprimrose cDNA library serving as template. PCR fragments of expectedsize may then be used to screen an evening primrose cDNA library.Hybridizing clones may then be sequenced and compared to the borage cDNAsequence to determine if the hybridizing clone represents an eveningprimrose Δ6-desatuase gene.

[0041] Transgenic organisms which gain the function of GLA production byintroduction of DNA encoding Δ6-desaturase also gain the function ofoctadecatetraeonic acid (18:4^(Δ6, 9,12 ,15)) production.Octadecatetraeonic acid is present normally in fish oils and in someplant species of the Boraginaceae family (Craig et al. [1964] J. Amer.Oil Chem. Soc. 41, 209-211; Gross et al. [1976] Can. J. Plant Sci. 56,659-664). In the transgenic organisms of the present invention,octadecatetraenoic acid results from further desaturation of a-linolenicacid by Δ6-desaturase or desaturation of GLA by Δ15-desaturase.

[0042] The 359 amino acids encoded by ORF2, i.e. the open reading frameencoding Synechocystis Δ6-desaturase, are shown as SEQ. ID NO:2. Theopen reading frame encoding the borage Δ6-desaturase is shown in SEQ IDNO: 5. The present invention further contemplates other nucleotidesequences which encode the amino acids of SEQ ID NO:2 and SEQ ID NO: 5.It is within the ken of the ordinarily skilled artisan to identify suchsequences which result, for example, from the degeneracy of the geneticcode. Furthermore, one of ordinary skill in the art can determine, bythe gain of function analysis described hereinabove, smallersubfragments of the fragments containing the open reading frames whichencode Δ6-desaturases.

[0043] The present invention contemplates any such polypeptide fragmentof Δ6-desaturase and the nucleic acids therefor which retain activityfor converting LA to GLA.

[0044] In another aspect of the present invention, a vector containing anucleic acid of the present invention or a smaller fragment containingthe promoter, coding sequence and termination region of a Δ6-desaturasegene is transferred into an organism, for example, cyanobacteria, inwhich the Δ6-desaturase promoter and termination regions are functional.Accordingly, organisms producing recombinant Δ6-desaturase are providedby this invention. Yet another aspect of this invention providesisolated Δ6-desaturase, which can be purified from the recombinantorganisms by standard methods of protein purification. (For example, seeAusubel et al. [1987] Current Protocols in Molecular Biology, GreenPublishing Associates, New York).

[0045] Vectors containing DNA encoding Δ6-desaturase are also providedby the present invention. It will be apparent to one of ordinary skillin the art that appropriate vectors can be constructed to direct theexpression of the Δ6-desaturase coding sequence in a variety oforganisms. Replicable expression vectors are particularly preferred.Replicable expression vectors as described herein are DNA or RNAmolecules engineered for controlled expression of a desired gene, i.e.the Δ6-desaturase gene. Preferably the vectors are plasmids,bacteriophages, cosmids or viruses. Shuttle vectors, e.g. as describedby Wolk et al. (1984) Proc. Natl. Acad. Sci. USA, 1561-1565 and Bustoset al. (1991) J. Bacteriol. 174, 7525-7533, are also contemplated inaccordance with the present invention. Sambrook et al. (1989), Goeddel,ed. (1990) Methods in Enzymology 185 Academic Press, and Perbal (1988) APractical Guide to Molecular Cloning, John Wiley and Sons, Inc., providedetailed reviews of vectors into which a nucleic acid encoding thepresent Δ6-desaturase can be inserted and expressed. Such vectors alsocontain nucleic acid sequences which can effect expression of nucleicacids encoding Δ6-desaturase. Sequence elements capable of effectingexpression of a gene product include promoters, enhancer elements,upstream activating sequences, transcription termination signals andpolyadenylation sites. The upstream 5′ untranslated region of theevening primrose Δ6-desaturase gene as depicted in FIG. 10 may also beused. Both constitutive and tissue specific promoters are contemplated.For transformation of plant cells, the cauliflower mosaic virus (CaMV)35S promoter, other constitutive promoters and promoters which areregulated during plant seed maturation are of particular interest. Allsuch promoter and transcriptional regulatory elements, singly or incombination, are contemplated for use in the present replicableexpression vectors and are known to one of ordinary skill in the art.The CaMV 355 promoter is described, for example, by Restrepo et al.(1990) Plant Cell 2, 987. Genetically engineered and mutated regulatorysequences are also contemplated.

[0046] The ordinarily skilled artisan can determine vectors andregulatory elements suitable for expression in a particular host cell.For example, a vector comprising the promoter from the gene encoding thecarboxylase of Anabaena operably linked to the coding region ofΔ6-desaturase and further operably linked to a termination signal fromSynechocystis is appropriate for expression of Δ6-desaturase incyanobacteria. “operably linked” in this context means that the promoterand terminator sequences effectively function to regulate transcription.As a further example, a vector appropriate for expression ofΔ6-desaturase in transgenic plants can comprise a seed specific promotersequence derived from helianthinin, napin, or glycinin operably linkedto the Δ6-desaturase coding region and further operably linked to a seedtermination signal or the nopaline synthase termination signal. As astill further example, a vector for use in expression of Δ6-desaturasein plants can comprise a constitutive promoter or a tissue specificpromoter operably linked to the Δ6-desaturase coding region and furtheroperably linked to a constitutive or tissue specific terminator or thenopaline synthase termination signal.

[0047] In particular, the helianthinin regulatory elements disclosed inapplicant's copending U.S. Application Ser. No. 682,354, filed Apr. 8,1991 and incorporated herein by reference, are contemplated as promoterelements to direct the expression of the Δ6-desaturases of the presentinvention. The albumin regulatory elements disclosed in applicant'scopending U.S. application Ser. No. 08/831,570 and the oleosinregulatory elements disclosed in applicant's copending U.S. applicationSer. No. 08/831,575 (both applications filed Apr. 9, 1997), andincorporated herein by reference, are also contemplated as elements todirect the expression of the Δ6-desaturases of the present invention.

[0048] Modifications of the nucleotide sequences or regulatory elementsdisclosed herein which maintain the functions contemplated herein arewithin the scope of this invention. Such modifications includeinsertions, substitutions and deletions, and specifically substitutionswhich reflect the degeneracy of the genetic code.

[0049] Standard techniques for the construction of such hybrid vectorsare well-known to those of ordinary skill in the art and can be found inreferences such as Sambrook et al. (1989), or any of the myriad oflaboratory manuals on recombinant DNA technology that are widelyavailable. A variety of strategies are available for ligating fragmentsof DNA, the choice of which depends on the nature of the termini of theDNA fragments. It is further contemplated in accordance with the presentinvention to include in the hybrid vectors other nucleotide sequenceelements which facilitate cloning, expression or processing, for examplesequences encoding signal peptides, a sequence encoding KDEL or relatedsequence, which is required for retention of proteins in the endoplasmicreticulum or sequences encoding transit peptides which directΔ6-desaturase to the chloroplast. Such sequences are known to one ofordinary skill in the art. An optimized transit peptide is described,for example, by Van den Broeck et al. (1985) Nature 313, 358.Prokaryotic and eukaryotic signal sequences are disclosed, for example,by Michaelis et al. (1982) Ann. Rev. Microbiol. 36, 425.

[0050] A further aspect of the instant invention provides organismsother than cyanobacteria or plants which contain the DNA encoding theΔ6-desaturase of the present invention. The transgenic organismscontemplated in accordance with the present invention include bacteria,cyanobacteria, fungi, and plants and animals. The isolated DNA of thepresent invention can be introduced into the host by methods known inthe art, for example infection, transfection, transformation ortransconjugation. Techniques for transferring the DNA of the presentinvention into such organisms are widely known and provided inreferences such as Sambrook et al. (1989).

[0051] A variety of plant transformation methods are known. TheΔ6-desaturase gene can be introduced into plants by a leaf disktransformation-regeneration procedure as described by Horsch et al.(1985) Science 227, 1229. Other methods of transformation, such asprotoplast culture (Horsch et al. (1984) Science 223, 496; DeBlock etal. (1984) EMBO J. 2, 2143; Barton et al. (1983) Cell 32, 1033) can alsobe used and are within the scope of this invention. In a preferredembodiment plants are transformed with Agrobacterium-derived vectorssuch as those described in Klett et al. (1987) Annu. Rev. Plant Physiol.38:467. However, other methods are available to insert the Δ6-desaturasegenes of the present invention into plant cells. Such alternativemethods include biolistic approaches (Klein et al. (1987) Nature 327,70), electroporation, chemically-induced DNA uptake, and use of virusesor pollen as vectors.

[0052] When necessary for the transformation method, the Δ6-desaturasegenes of the present invention can be inserted into a planttransformation vector, e.g. the binary vector described by Bevan (1984)Nucleic Acids Res. 12, 8111. Plant transformation vectors can be derivedby modifying the natural gene transfer system of Agrobacteriumtumefaciens. The natural system comprises large Ti(tumor-inducing)-plasmids containing a large segment, known as T-DNA,which is transferred to transformed plants. Another segment of the Tiplasmid, the vir region, is responsible for T-DNA transfer. The T-DNAregion is bordered by terminal repeats. In the modified binary vectorsthe tumor-inducing genes have been deleted and the functions of the virregion are utilized to transfer foreign DNA bordered by the T-DNA bordersequences. The T-region also contains a selectable marker for antibioticresistance, and a multiple cloning site for inserting sequences fortransfer. Such engineered strains are known as “disarmed” A. tumefaciensstrains, and allow the efficient transformation of sequences bordered bythe T-region into the nuclear genomes of plants.

[0053] Surface-sterilized leaf disks are inoculated with the “disarmed”foreign DNA-containing A. tumefaciens, cultured for two days, and thentransferred to antibiotic-containing medium. Transformed shoots areselected after rooting in medium containing the appropriate antibiotic,transferred to soil and regenerated.

[0054] Another aspect of the present invention provides transgenicplants or progeny of these plants containing the isolated DNA of theinvention. Both monocotyledenous and dicotyledenous plants arecontemplated. Plant cells are transformed with the isolated DNA encodingΔ6-desaturase by any of the plant transformation methods describedabove. The transformed plant cell, usually in a callus culture or leafdisk, is regenerated into a complete transgenic plant by methodswell-known to one of ordinary skill in the art (e.g. Horsch et al.(1985) Science 227, 1129). In a preferred embodiment, the transgenicplant is sunflower, oil seed rape, maize, tobacco, peanut or soybean.Since progeny of transformed plants inherit the DNA encodingΔ6-desaturase, seeds or cuttings from transformed plants are used tomaintain the transgenic plant line.

[0055] The present invention further provides a method for providingtransgenic plants with an increased content of GLA. This method includesintroducing DNA encoding Δ6-desaturase into plant cells which lack orhave low levels of GLA but contain LA, and regenerating plants withincreased GLA content from the transgenic cells. In particular,commercially grown crop plants are contemplated as the transgenicorganism, including, but not limited to, sunflower, soybean, oil seedrape, maize, peanut and tobacco.

[0056] The present invention further provides a method for providingtransgenic organisms which contain GLA. This method comprisesintroducing DNA encoding Δ6-desaturase into an organism which lacks orhas low levels of GLA, but contains LA. In another embodiment, themethod comprises introducing one or more expression vectors whichcomprise DNA encoding Δ12-desaturase and Δ6-desaturase into organismswhich are deficient in both GLA and LA. Accordingly, organisms deficientin both LA and GLA are induced to produce LA by the expression ofΔ12-desaturase, and GLA is then generated due to the expression ofΔ6-desaturase. Expression vectors comprising DNA encodingΔ12-desaturase, or Δ12-desaturase and Δ6-desaturase, can be constructedby methods of recombinant technology known to one of ordinary skill inthe art (Sambrook et al., 1989) and the published sequence ofΔ12-desaturase (Wada et al [1990] Nature (London) 347, 200-203. Inaddition, it has been discovered in accordance with the presentinvention that nucleotides 2002-3081 of SEQ. ID NO:1 encodecyanobacterial Δ12-desaturase. Accordingly, this sequence can be used toconstruct the subject expression vectors. In particular, commerciallygrown crop plants are contemplated as the transgenic organism,including, but not limited to, sunflower, soybean, oil seed rape, maize,peanut and tobacco.

[0057] The present invention is further directed to a method of inducingchilling tolerance in plants. Chilling sensitivity may be due to phasetransition of lipids in cell membranes. Phase transition temperaturedepends upon the degree of unsaturation of fatty acids in membranelipids, and thus increasing the degree of unsaturation, for example byintroducing Δ6-desaturase to convert LA to GLA, can induce or improvechilling resistance. Accordingly, the present method comprisesintroducing DNA encoding Δ6-desaturase into a plant cell, andregenerating a plant with improved chilling resistance from saidtransformed plant cell. In a preferred embodiment, the plant is asunflower, soybean, oil seed rape, maize, peanut or tobacco plant.

[0058] The following examples further illustrate the present invention.

EXAMPLE 1 Strains and Culture Conditions

[0059] Synechocystis (PCC 6803, ATCC 27184), Anabaena (PCC 7120, ATCC27893) and Synechococcus (PCC 7942, ATCC 33912) were grownphotoautotrophically at 30° C. in BG11N+ medium (Rippka et al. [1979] J.Gen. Microbiol. 111, 1-61) under illumination of incandescent lamps (60μE.m⁻².S⁻). Cosmids and plasmids were selected and propagated inEscherichia coli strain DH5a on LB medium supplemented with antibioticsat standard concentrations as described by Maniatis al . (1982)Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory,Cold Spring, N.Y.

EXAMPLE 2 Construction of Synechocystis Cosmid Genomic Library

[0060] Total genomic DNA from Synechocystis (PCC 6803) was partiallydigested with Sau3A and fractionated on a sucrose gradient (Ausubel etal. [1987] Current Protocols in Molecular Biology, Greene PublishingAssociates and Wiley Interscience, New York). Fractions containing 30 to40 kb DNA fragments were selected and ligated into the dephosphorylatedBamHI site of the cosmid vector, pDUCΔ7 (Buikema et al. [1991] J.Bacteriol. 173, 1879-1885). The ligated DNA was packaged in vitro asdescribed by Ausubel et al. (1987), and packaged phage were propagatedin E. coli DH5α containing the AvaI and Eco4711 methylase helperplasmid, pRL528 as described by Buikema et al. (1991). A total of 1152colonies were isolated randomly and maintained individually in twelve96-well microtiter plates.

EXAMPLE 3 Gain-of-Function Expression of GLA in Anabaena

[0061] Anabaena (PCC 7120), a filamentous cyanobacterium, is deficientin GLA but contains significant amounts of linoleic acid, the precursorfor GLA (FIG. 2; Table 2). The Synechocystis cosmid library described inExample 2 was conjugated into Anabaena (PCC 7120) to identifytransconjugants that produce GLA. Anabaena cells were grown to mid-logphase in BG11N+ liquid medium and resuspended in the same medium to afinal concentration of approximately 2×10⁸ cells per ml. A mid-log phaseculture of E. coli RP4 (Burkardt et al. [1979] J. Gen. Microbiol. 114,341-348) grown in LB containing ampicillin was washed and resuspended infresh LB medium. Anabaena and RP4 were then mixed and spread evenly onBG11N+ plates containing 5% LB. The cosmid genomic library was replicaplated onto LB plates containing 50 μg/ml kanamycin and 17.5 μg/mlchloramphenicol and was subsequently patched onto BG11N+ platescontaining Anabaena and RP4. After 24 hours of incubation at 30° C., 30μg/ml of neomycin was underlaid; and incubation at 30° C. was continueduntil transconjugants appeared.

[0062] Individual transconjugants were isolated after conjugation andgrown in 2 ml BG11N+ liquid medium with 15 μg/ml neomycin. Fatty acidmethyl esters were prepared from wild type cultures and culturescontaining pools of ten transconjugants as follows. Wild type andtransgenic cyanobacterial cultures were harvested by centrifugation andwashed twice with distilled water. Fatty acid methyl esters wereextracted from these cultures as described by Dahmer et al. (1989) J.Amer. Oil. Chem. Soc. 66, 543-548 and were analyzed by Gas LiquidChromatography (GLC) using a Tracor-560 equipped with a hydrogen flameionization detector and capillary column (30 m×0.25 mm bonded FSOTSuperox II, Alltech Associates Inc., IL). Retention times andco-chromatography of standards (obtained from Sigma Chemical Co.) wereused for identification of fatty acids. The average fatty acidcomposition was determined as the ratio of peak area of each C18 fattyacid normalized to an internal standard.

[0063] Representative GLC profiles are shown in FIG. 2. C18 fatty acidmethyl esters are shown. Peaks were identified by comparing the elutiontimes with known standards of fatty acid methyl esters and wereconfirmed by gas chromatography-mass spectrometry. Panel A depicts GLCanalysis of fatty acids of wild type Anabaena. The arrow indicates themigration time of GLA. Panel B is a GLC profile of fatty acids oftransconjugants of Anabaena with pAM542+1.8F. Two GLA producing pools(of 25 pools representing 250 transconjugants) were identified thatproduced GLA. Individual transconjugants of each GLA positive pool wereanalyzed for GLA production; two independent transconjugants, AS13 andAS75, one from each pool, were identified which expressed significantlevels of GLA and which contained cosmids, cSy13 and cSy75, respectively(FIG. 3). The cosmids overlap in a region approximately 7.5 kb inlength. A 3.5 kb NheI fragment of cSy75 was recloned in the vectorpDUCA7 and transferred to Anabaena resulting in gain-of-functionexpression of GLA (Table 2).

[0064] Two NheI/Hind III subfragments (1.8 and 1.7 kb) of the 3.5 kb NheI fragment of cSy75-3.5 were subcloned into “pBLUESCRIPT” (Stratagene)(FIG. 3) for sequencing. Standard molecular biology techniques wereperformed as described by Maniatis et al. (1982) and Ausubel et al.(1987). Dideoxy sequencing (Sanger et al. [1977] Proc. Natl. Acad. Sci.USA 74, 5463-5467) of pBS1.8 was performed with “SEQUENASE” (UnitedStates Biochemical) on both strands by using specific oligonucleotideprimers synthesized by the Advanced DNA Technologies Laboratory (BiologyDepartment, Texas A & M University). DNA sequence analysis was done withthe GCG (Madison, Wis.) software as described by Devereux et al. (1984)Nucleic Acids Res. 12, 387-395.

[0065] Both NheI/HindIII subfragments were transferred into a conjugalexpression vector, AM542, in both forward and reverse orientations withrespect to a cyanobacterial carboxylase promoter and were introducedinto Anabaena by conjugation. Transconjugants containing the 1.8 kbfragment in the forward orientation (AM542-1.8F) produced significantquantities of GLA and octadecatetraenoic acid (FIG. 2; Table 2).Transconjugants containing other constructs, either reverse oriented 1.8kb fragment or forward and reverse oriented 1.7 kb fragment, did notproduce detectable levels of GLA (Table 2).

[0066]FIG. 2 compares the C18 fatty acid profile of an extract from wildtype Anabaena (FIG. 2A) with that of transgenic Anabaena containing the1.8 kb fragment of cSy75-3.5 in the forward orientation (FIG. 2B). GLCanalysis of fatty acid methyl esters from AM542-1.8F revealed a peakwith a retention time identical to that of authentic GLA standard.Analysis of this peak by gas chromatography-mass spectrometry (GC-MS)confirmed that it had the same mass fragmentation pattern as a GLAreference sample. Transgenic Anabaena with altered levels ofpolyunsaturated fatty acids were similar to wild type in growth rate andmorphology.

EXAMPLE 4 Transformation of Synechococcus With Δ6 and Δ12 DesaturaseGenes

[0067] A third cosmid, cSy7, which contains a Δ12-desaturase gene, wasisolated by screening the Synechocystis genomic library with aoligonucleotide synthesized from the published SynechocystisΔ12-desaturase gene sequence (Wada et al. [1990] Nature (London) 347,200-203). A 1.7 kb AvaI fragment from this cosmid containing theΔ12-desaturase gene was identified and used as a probe to demonstratethat cSy13 not only contains a Δ6-desaturase gene but also aΔ12-desaturase gene (FIG. 3). Genomic Southern blot analysis furthershowed that both the Δ6-and Δ12-desaturase genes are unique in theSynechocystis genome so that both functional genes involved in C18 fattyacid desaturation are linked closely in the Synechocystis genome.

[0068] The unicellular cyanobacterium Synechococcus (PCC 7942) isdeficient in both linoleic acid and GLA(3). The Δ12 and Δ6-desaturasegenes were cloned individually and together into pAM854 (Bustos et al.[1991] J. Bacteriol. 174, 7525-7533), a shuttle vector that containssequences necessary for the integration of foreign DNA into the genomeof Synechococcus (Golden et al. [1987] Methods in Enzymol. 153,215-231). Synechococcus was transformed with these gene constructs andcolonies were selected. Fatty acid methyl esters were extracted fromtransgenic Synechococcus and analyzed by GLC.

[0069] Table 2 shows that the principal fatty acids of wild typeSynechococcus are stearic acid (18:0) and oleic acid (18:1).Synechococcus transformed with pAM854-Δ12 expressed linoleic acid (18:2)in addition to the principal fatty acids. Transformants with pAM854-Δ6and Δ12 produced both linoleate and GLA (Table 1). These resultsindicated that Synechococcus containing both Δ12- and Δ6-desaturasegenes had gained the capability of introducing a second double bond atthe Δ12 position and a third double bond at the AG position of C18 fattyacids. However, no changes in fatty acid composition was observed in thetransformant containing pAM854-Δ6, indicating that in the absence ofsubstrate synthesized by the Δ12 desaturase, the AG-desaturase isinactive. This experiment further confirms that the 1.8 kb NheI/HindIIIfragment (FIG. 3) contains both coding and promoter regions of theSynechocystis Δ6-desaturase gene. Transgenic Synechococcus with alteredlevels of polyunsaturated fatty acids were similar to wild type ingrowth rate and morphology. TABLE 2 Composition of C18 Fatty Acids inWild Type and Transgenic Cyanobacteria Fatty acid (%) Strain 18:0 18:118:2 18:3(α) 18:3(γ) 18:4 Wild Type Syacchocystis (sp. 13.6 4.5 54.5 —27.3 — PCC6803) Anabaena (sp. 2.9 24.8 37.1 35.2 — — PCC7120)Syacchococcus (sp. 20.6 79.4 — — — — PCC7942) Anabaena Transcon- Agentsy75 3.8 24.4 22.3 9.1 27.9 123 75-3.5 4.3 27.6 18.1 3.2 40.4 6.4pAM542 - 1.8 F 4.2 13.9 12.1 19.1 25.4 25.4 pAM542 - 1.8 F 7.7 23.1 38.430.8 — — pAM542 - 1.7 F 2.8 27.8 36.1 33.3 — — pAM542 - 1.7 R 2.8 25.442.3 29.6 — — Syaschococcus Trans- ants pAM854 27.8 72.2 — — — —pAM854 - Δ¹² 4.0 43.2 46.0 — — — pAM854 - Δ⁶ 18.2 81.8 — — — — pAM854 -Δ⁶ & Δ¹² 42.7 25.3 19.5 — 16.5 —

EXAMPLE 5 Nucleotide Sequence of Δ6-Desaturase

[0070] The nucleotide sequence of the 1.8 kb fragment of cSy75-3.5including the functional Δ6-desaturase gene was determined. An openreading frame encoding a polypeptide of 359 amino acids was identified(FIG. 4). A Kyte-Doolittle hydropathy analysis (Kyte et al. [1982] J.Mol. Biol. 157, 105-132) identified two regions of hydrophobic aminoacids that could represent transmembrane domains (FIG. 1A); furthermore,the hydropathic profile of the Δ6-desaturase is similar to that of theΔ12-desaturase gene (FIG. 1B; Wada et al.) and Δ9-desaturases (Thiede etal. [1986] J. Biol. Chem. 261, 13230-13235). However, the sequencesimilarity between the Synechocystis Δ6- and Δ12-desaturases is lessthan 40% at the nucleotide level and approximately 18% at the amino acidlevel.

EXAMPLE 6 Transfer of Cyanobacterial Δ⁶-Desaturase into Tobacco

[0071] The cyanobacterial Δ⁶-desaturase gene was mobilized into a plantexpression vector and transferred to tobacco using Agrobacteriummediated gene transfer techniques. To ensure that the transferreddesaturase is appropriately expressed in leaves and developing seeds andthat the desaturase gene product is targeted to the endoplasmicreticulum or the chloroplast, various expression cassettes withSynechocystis Δ-desaturase open reading frame (ORF) were constructed.Components of these cassettes include: (i) a 35S promoter or seedspecific promoter derived from the sunflower helianthinin gene to driveΔ6-desaturase gene expression in all plant tissues or only in developingseeds respectively, (ii) a putative signal peptide either from carrotextension gene or sunflower helianthinin gene to target newlysynthesized Δ6-desaturase into the ER, (iii) an ER lumen retentionsignal sequence (KDEL) at the COOH-terminal of the Δ6-desaturase ORF,and (iv) an optimized transit peptide to target Δ6 desaturase into thechloroplast. The 35S promoter is a derivative of pRTL2 described byRestrepo et al. (1990). The optimized transit peptide sequence isdescribed by Van de Broeck et al. (1985). The carrot extensin signalpeptide is described by Chen et al (1985) EMBO J. 9, 2145.

[0072] Transgenic tobacco plants were produced containing a chimericcyanobacterial desaturase gene, comprised of the SynechocystisΔ6-desaturase gene fused to an endoplasmic reticulum retention sequence(KDEL) and extensin signal peptide driven by the CaMV 35S promoter. PCRamplifications of transgenic tobacco genomic DNA indicate that theΔ6-desaturase gene was incorporated into the tobacco genome. Fatty acidmethyl esters of leaves of these transgenic tobacco plants wereextracted and analyzed by Gas Liquid Chromatography (GLC). Thesetransgenic tobacco accumulated significant amounts of GLA (FIG. 4). FIG.4 shows fatty acid methyl esters as determined by GLC. Peaks wereidentified by comparing the elution times with known standards of fattyacid methyl ester. Accordingly, cyanobacterial genes involved in fattyacid metabolism can be used to generate transgenic plants with alteredfatty acid compositions.

EXAMPLE 7 Construction of Borage CDNA Library

[0073] Membrane bound polysomes were isolated from borage seeds 12 dayspost pollination (12 DPP) using the protocol established for peas byLarkins and Davies (1975 Plant Phys. 55:749-756). RNA was extracted fromthe polysomes as described by Mechler (1987 Methods in Enzymology152:241-248, Academic Press).

[0074] Poly-A+ RNA was isolated from the membrane bound polysomal RNA byuse of Oligotex-dT beads (Qiagen). Corresponding cDNA was made usingStratagene's ZAP cDNA synthesis kit. The cDNA library was constructed inthe lambda ZAP II vector (Stratagene) using the lambda ZAP II vectorkit. The primary library was packaged in Gigapack II Gold packagingextract (Stratagene). The library was used to generate expressedsequence tags (ESTs), and sequences corresponding to the tags were usedto scan the GenBank database.

EXAMPLE 8 Hybridization Protocol

[0075] Hybridization probes for screening the borage CDNA library weregenerated by using random primed DNA synthesis as described by Ausubelet al (1994 Current Protocols in Molecular Biology, Wiley Interscience,N.Y.) and corresponded to previously identified abundantly expressedseed storage protein cDNAs. Unincorporated nucleotides were removed byuse of a G-50 spin column (Boehringer Manheim). Probe was denatured forhybridization by boiling in a water bath for 5 minutes, then quicklycooled on ice. Filters for hybridization were prehybridized at 60° C.for 2-4 hours in prehybridization solution (6XSSC [Maniatis et al 1984Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory],1× Denharts Solution, 0.05% sodium pyrophosphate, 100 μg/ml denaturedsalmon sperm DNA). Denatured probe was added to the hybridizationsolution (6× SSC, 1× Denharts solution, 0.05% sodium pyrophosphate, 100μg/ml denatured salmon sperm DNA) and incubated at 60° C. with agitationovernight. Filters were washed in 4×, 2×, and 1× SET washes for 15minutes each at 60° C. A 20× SET stock solution is 3 M NaCl, 0.4 M Trisbase, 20 mM Na₂EDTA-2H₂O. The 4× SET wash was 4× SET, 12.5 mM PO₄, pH6.8 and 0.2% SDS. The 2× SET wash was 2× SET, 12.5 mM PO₄, pH 6.8 and0.2% SDS. The 1× SET wash was 133 SET, 12.5 mM PO₄, pH 6.8 and 0.2% SDS.Filters were allowed to air dry and were then exposed to X-ray film for24 hours with intensifying screens at 80° C.

EXAMPLE 9 Random Sequencing of cDNAs from a Borage Seed (12 DPP)Membrane-bound Polysomal Library

[0076] The borage cDNA library was plated at low density (500 pfu on 150mm petri dishes). Highly prevalent seed storage protein cDNAs were“subtracted” by screening with the previously identified correspondingcDNAs. Non-hybridizing plaques were excised using Stratagene's excisionprotocol and reagents. Resulting bacterial colonies were used toinoculate liquid cultures and were either sequenced manually or by anABI automated sequencer. Each cDNA was sequenced once and a sequence taggenerated from 200-300 base pairs. All sequencing was performed by cyclesequencing (Epicentre). Over 300 ESTs were generated. Each sequence tagwas compared to GenBank database by BLASTX computer program and a numberof lipid metabolism genes, including the Δ6-desaturase were identified.

[0077] Database searches with a cDNA clone designated mbp-65 usingBLASTX with the GenBank database resulted in a significant match to theSynechocystis Δ6-desaturase. It was determined however, that this clonewas not a full length cDNA. A full length cDNA was isolated using mbp-65to screen the borage membrane-bound polysomal library. The sequence ofthe isolated cDNA was determined (FIG. 5A, SEQ ID NO:4) and the proteinsequence of the open reading frame (FIG. 5B, SEQ ID NO:5) was comparedto other known desaturases using Geneworks (IntelligGenetics) proteinalignment program (FIG. 2). This alignment indicated that the cDNA wasthe borage Δ6-desaturase gene.

[0078] Although similar to other known plant desaturases, the boragedelta 6-desaturase is distinct as indicated in the dendrogram shown inFIG. 6. Furthermore, comparison of the amino acid sequencescharacteristic of desaturases, particularly those proposed to beinvolved in metal binding (metal box 1 and metal box 2), illustrates thedifferences between the borage delta 6-desaturase and other plantdesaturases (Table 3).

[0079] The borage delta 6-desaturase is distinguished from thecyanobacterial form not only in over all sequence (FIG. 6) but also inthe lipid box, metal box 1 and metal box 2 amino acid motifs (Table 3).As Table 3 indicates, all three motifs are novel in sequence. Only theborage delta 6-desaturase metal box 2 showed some relationship to theSynechocystis delta-6 desaturase metal box 2.

[0080] In addition, the borage delta 6-desaturase is also distinct fromanother borage desaturase gene, the delta-12 desaturase. P1-81 is a fulllength cDNA that was identified by EST analysis and shows highsimilarity to the Arabidopsis delta-12 desaturase (Fad 2). A comparisonof the lipid box, metal box 1 and metal box 2 amino acid motifs (Table3) in borage delta 6 and delta-12 desaturases indicates that littlehomology exists in these regions. The placement of the two sequences inthe dendrogram in FIG. 6 indicates how distantly related these two genesare. TABLE 3 Comparison of common amino acid motifs in membrane-bounddesaturases Amino Acid Motif Desaturase Lipid Box Metal Box 1 Metal Box2 Borage Δ⁶ WIGHDAGH (SEQ. ID. NO: 6) HNAHH  (SEQ. ID. NO: 12) FQIEHH(SEQ. ID. NO: 20) Synechocystis Δ⁶ NVGHDANH (SEQ. ID. NO: 7) HNYLHH(SEQ. ID. NO: 13) HQVTHH (SEQ. ID. NO: 21) Arab. chloroplast Δ¹⁵VLGHDCGH (SEQ. ID. NO: 8) HRTHH  (SEQ. ID. NO: 14) HVIHH  (SEQ. ID. NO:22) Rice Δ¹⁵ VLGHDCGH (SEQ. ID. NO: 8) HRTHH  (SEQ. ID. NO: 14)HVIHH  (SEQ. ID. NO: 22) Glycine chloroplast Δ¹⁵ VLGHDCGH (SEQ. ID. NO:8) HRTHH  (SEQ. ID. NO: 14) HVIHH  (SEQ. ID. NO: 22) Arab. fad3 (Δ¹⁵)VLGHDCGH (SEQ. ID. NO: 8) HRTHH  (SEQ. ID. NO: 14) HVIHH  (SEQ. ID. NO:22) Brassica fad3 (Δ¹⁵) VLGHDCGH (SEQ. ID. NO: 8) HRTHH  (SEQ. ID. NO:14) HVIHH  (SEQ. ID. NO: 22) Borage Δ¹² (P1-81)* VIAHECGH (SEQ. ID. NO:9) HRRHN  (SEQ. ID. NO: 15) HVANH  (SEQ. ID. NO: 23) Arab. fad2 (Δ¹² )VIAHECGH (SEQ. ID. NO: 9) HRRHH  (SEQ. ID. NO: 15) HVAHH  (SEQ. ID. NO:23) Arab. chloroplast Δ¹² VIGHDCAH (SEQ. ID. NO: 10) HDRHH  (SEQ. ID.NO: 16) HIPHH  (SEQ. ID. NO: 24) Glycine plastid Δ¹² VIGHDCAH (SEQ. ID.NO: 10) HDRHH  (SEQ. ID. NO: 16) HIPHH  (SEQ. ID. NO: 24) Spinachplastidial n-6 VIGHDCAH (SEQ. ID. NO: 10) HDQHH  (SEQ. ID. NO: 17)HIPHH  (SEQ. ID. NO: 24) Synechocystis Δ¹² VVGHDCGH (SEQ. ID. NO: 11)HDHHH  (SEQ. ID. NO: 18) HIPHH  (SEQ. ID. NO: 24) Anabaena Δ¹² VLGHDCGH(SEQ. ID. NO: 8) HNHHH  (SEQ. ID. NO: 19) HVPHH  (SEQ. ID. NO: 25)

EXAMPLE 10 Construction of 222.1Δ⁶NOS for transient and expression

[0081] The vector pBI221 (Jefferson et al. 1987 EMBO J. 6:3901-3907) wasprepared for ligation by digestion with BamHI and EcoICR I (Promega)which excises the GUS coding region leaving the 35S promoter and NOSterminator intact. The borage Δ6-desaturase cDNA was excised from theBluescript plasmid (Stratagene) by digestion with BamHI and XhoI. TheXhoI end was made blunt by use of the Klenow fragment. This fragment wasthen cloned into the BamHI/EcoICR I sites of pBI221, yielding 221.1Δ⁶NOS(FIG. 7). In 221.1Δ.NOS, the remaining portion (backbone) of therestriction map depicted in FIG. 7 is pBI221.

EXAMPLE 11 Construction of 121.1Δ⁶.NOS for stable transformation

[0082] The vector pBI121 (Jefferson et al. 1987 EMBO J. 6:3901-3907) wasprepared for ligation by digestion with BamHI and EcoICR I (Promega)which excises the GUS coding region leaving the 35S promoter and NOSterminator intact. The borage Δ6-desaturase cDNA was excised from theBluescript plasmid (Stratagene) by digestion with BamHI and XhoI. TheXhoI end was made blunt by use of the Klenow fragment. This fragment wasthen cloned into the BamHI/EcoICR I sites of pBI121, yielding 121.1Δ⁶NOS(FIG. 7). In 121.Δ⁶.NOS, the remaining portion (backbone) of therestriction map depicted in FIG. 7 is pBI121.

EXAMPLE 12 Transient Expression

[0083] All work involving protoplasts was performed in a sterile hood.One ml of packed carrot suspension cells were digested in 30 mlsplasmolyzing solution (25 g/l KC1, 3.5 g/l CaCl₂—H₂O, 10 mM MES, pH 5.6and 0.2 M mannitol) with 1% cellulase, 0.1% pectolyase, and 0.1%dreisalase overnight, in the dark, at room temperature. Releasedprotoplasts were filtered through a 150 μm mesh and pelleted bycentrifugation (100× g, 5 min.) then washed twice in plasmolyzingsolution. Protoplasts were counted using a double chamberedhemocytometer. DNA was transfected into the protoplasts by PEG treatmentas described by Nunberg and Thomas (1993 Methods in Plant MolecularBiology and Biotechnology, B. R. Glick and J. E. Thompson, eds. pp.241-248) using 10⁶ protoplasts and 50-70 μg of plasmid DNA (221.Δ6.NOS).Protoplasts were cultured in 5 mls of MS media supplemented with 0.2 Mmannitol and 3 μm 2,4-D for 48 hours in the dark with shaking.

EXAMPLE 13 Stable Transformation of Tobacco

[0084] 121.1Δ⁶NOS plasmid construction was used to transform tobacco(Nicotiana tabacum cv. xanthi) via Agrobacterium according to standardprocedures (Horsh et al., 1985 Science 221: 1229-1231; Bogue et al.,1990 Mol. Gen. Genet. 221:49-57), except that initial transformants wereselected on 100 μg/ml kanamycin.

EXAMPLE 14 Preparation and Analysis of Fatty Acid Methyl Esters (FAMEs)

[0085] Tissue from transfected protoplasts and transformed tobaccoplants was frozen in liquid nitrogen and lyophilized overnight. FAMEswere prepared as described by Dahmer et al (1989 J. Amer. Oil Chem. Soc.66-548). In some cases, the solvent was evaporated again, and the FAMEswere resuspended in ethyl acetate and extracted once with deionizedwater to remove any water soluble contaminants. The FAMEs were analyzedby gas chromatography (GC) on a J&W Scientific DB-wax column (30 mlength, 0.25 mm ID, 0.25 um film).

[0086] An example of a transient assay is shown in FIG. 8 whichrepresents three independent transfections pooled together. The additionof the borage Δ6-desaturase cDNA corresponds with the appearance ofgamma linolenic acid (GLA) which is one of the possible products ofΔ6-desaturase. Furthermore, transgenic tobacco containing the borageΔ6-desaturase driven by the cauliflower mosaic virus 35S promoter alsoproduce GLA as well as octa-decaenoic acid (18:4) which is formed by thefurther desaturation of GLA (FIG. 9). These results indicate that theborage delta 6-desaturase gene can be used to transform plant cells toachieve altered fatty acid compositions.

EXAMPLE 15 Isolation of an Evening Primrose Δ6-desaturase Gene

[0087] Total RNA was isolated from evening primrose embryos using themethod of Chang, Puryear, and Cairney (1993) Plant Mol Biol Reporter11:113-116. Poly A⁺RNA was selected on oligotex beads (Qiagen) and usedas a template for cDNA synthesis. The CDNA library was constructed inthe lambda ZAP II vector (Stratagene) using the lambda ZAP II vectorkit. The primary library was packaged with Gigapack II Gold packagingextract (Stratagene).

[0088] PCR primers based on sequences in the borage Δ6-desaturase genewere synthesized by a commercial source using standard protocols andincluded the following oligonucleotides:

[0089] 5′ AAACCAATCCATCCAAGRA 3′ SEQ ID NO:27

[0090] 5′ KTGGTGGAAATGGAMSCATAA 3′ SEQ ID NO:28

[0091] (R=A and G, K=G and T, M=A and C, S=G and C)

[0092] A primer that matches a region that flanks the insertion site ofthe lambda ZAP II vector was also synthesized using an ABI394 DNAsynthesizer and standard protocols. This primer ahd the followingsequence:

[0093] 5′ TCTAGAACTAGTGGATC 3′ SEQ ID NO:29

[0094] An aliquot of the cDNA library was used directly as template in aPCR reaction using SEQ ID NO: 27 and SEQ ID NO:29 as primers. Thereactions were carried out in a volume of 50 μl using an annealingtemperature of 50° C. for 2 minutes, an extension temperature of 72° C.for 1.5 minutes, and a melting temperature of 94° C. for 1 minute for 29cycles. A final cycle with a 2 minute annealing at 50° C. and a 5 minuteextension at 72° C. completed the reaction. One μl from this reactionwas used as a template in a second reaction using the same conditionsexcept that the primers were SEQ ID NO:27 and SEQ ID NO:28. A DNAfragment of predicted size based on the location of the primer sequencesin the the borage Δ6-desaturase cDNA was isolated.

[0095] This PCR fragment was cloned into pT7 Blue (Novagen) and used toscreen the evening primrose cDNA library at low stringency conditions:The hybridization buffer used was 1% bovine serum albumin (crystallinefraction V), 1 mM EDTA, 0.5 M NaHPO₄pH7.2, and 7% SDS. Thehybridizations were at 65 C. The wash buffer was 1 mM Na₂EDTA, 40 mMNaHPO₄pH7.2 and 1% SDS. Primary screens were washed at 25° C. Secondaryand tertiary screens were washed at 25° C., 37° C., and 42° C. One ofthe positively hybridizing clones that was identified in the eveningprimrose cDNA library was excised as a phagemid in pBluescript. The DNAsequence of the 1687 bp insert of this phagemid (pIB9748-4) wasdetermined (FIG. 10, SEQ ID NO: 26) using the ABIPRISM™ dye terminatorcycle sequencing core kit from Perkin Elmer according to themanufacturer's protocol. The sequence encodes a full length protein of450 amino acids (SEQ ID NO:27) with a molecular weight of 51492 daltons.

[0096] Alignment of the deduced amino acid sequence with that of borageΔ6-desaturase was performed using the Geneworks program (FIG. 11). Theevening primrose Δ6-desaturase protein is identical at 58% of theresidues and similar at an additional 20% of the residues. Only twosmall gaps, near the carboxy terminal end of the protein were introducedby the program to obtain the alignment (FIG. 11). The two proteins werecompared using two different alogorithms that measure the hydrophobicityof regions to the protein. FIGS. 12A and 12B are Kyte-Doolittlehydrophobicity plots of borage Δ6-desaturase and evening primroseΔ6-desaturase, respectively. FIGS. 13A and 13B are Hopwoodhydrophobicity plots generated in the program DNA Strider for the sameproteins. A discussion of the algorithim used to generate these plotscan be found in Hopp, T. P. and Woods, K. R. 1983 Molecular Immunology20:483-89. Substantial similarity exists between the borage and eveningprimrose proteins using either algorithm. TMPredict, a program thatpredicts'the location of transmembrane regions of proteins was run onthe two sequences and again similar results were obtained (FIGS. 14 and15). Several weights matrices are used in scoring the predictions asreported in Hofmann, K. and Stoffel, W. 1993 Biol. C. Hoppe-Scyler347:156. Positive values (x-axis) greater than 500 are consideredsignificant predictors of a membrane spanning region; the x-axisrepresents the linear amino acid sequences.

[0097] The membrane bound desaturases of plants possess three histidinerich motiffs (HRMs). These motiffs are identified in the eveningprimrose sequence and are indicated in FIG. 10 by underlined plain text.The motifs in this sequence were identical to those found in borageΔ6-desaturase with the exception of those that are italicized (S 161 andL374). The borage Δ6-desaturase is unique among known membrane bounddesaturases in having a cytochrome b5 domain at the carboxy terminalend. The evening primrose protein encoded by pIB9748-4 also has thisdomain. The heme binding motiff of chtochrome b5 proteins is indicatedin FIG. 10 by underlined bold text.

[0098] These data indicate that a Δ6-desaturase cDNA from eveningprimrose has been isolated and characterized.

EXAMPLE 16 Construction of Expression Vectors for Transient and StableExpression of an Evening Primrose Δ6-desaturase

[0099] The evening primrose Δ6-desaturase cDNA is excised from theBluescript phagemid by digestion with Xba I and Xho I. The entire cDNAsequence including the 5′ transcribed but untranslated region depictedin FIG. 10 (SEQ ID NO:26) is operably linked to any one of variouspromoters and/or other regulatory elements in an expression vector, inorder to effect transcription and translation of the Δ6-desaturase gene.Alternatively, the cDNA sequence depicted in FIG. 10 may be trimmed atthe 5θ end so that the 5′ transcribed but untranslated sequence isremoved. The A of the ATG translational start codon is then made thefirst nucleotide following the promoter and/or other regulatory sequencein an expression vector.

[0100] In order to express the subject evening primrose cDNA in pBI221(Jefferson et al. 1987 EMBO J. 6:3901-3907) the following manipulationsare performed:

[0101] The plasmid pBI221 is digested with EcoICR I (Promega) or Ecl 136II (NEB) and Xba I which excises the GUS coding region and leaves the35S promoter and NOS terminator intact. The evening primroseΔ6-desaturase cDNA is excised from pIB9748-4 by digestion with Xba I andXho I. The Xho I end is made blunt by use of the Klenow fragment. Theexcised gene is then cloned into the cloned into the Xba I/Eco ICR Isites of pBI221. The resulting construct is then transfected into carrotprotoplasts. One ml of packed carrot suspension cells are digested in 30ml of plasmolyzing solution (25 g/l KCl 3.5 g/l CaCl₂-H₂O, 10 mM MES, pH5.6 and 0.2 M mannitol) with 1% cellulase 0.1% pectolyase, and 0.1%dreisalase overnight, in the dark, at room temperature. Releasedprotoplasts are filtered through a 150 μm mesh and pelleted bycentrifugation (100 × g, 5 minutes), then washed twice in plasmolyzingsolution. Protoplasts are counted using a double chamberedhemocytometer. DNA is transfected into the protoplasts by PEG treatmentas described by Nunberg and Thomas (1993 Methods in Plant MolecularBiology and Biotechnology, B. R. Glick and J. E. Thompson, eds. pp241-248) using 106 protoplasts and 50-70 ug of DNA from the aboveconstruct. Protoplasts are cultured in 5 ml of MS medium supplementedwith 0.2 M mannitol and 3 μM 2, 4-D for 48 hours in the dark withshaking. Tobacco is transformed with the same Δ6-desaturase expressionconstruct by following the method of Example 13.

[0102] In order to express the subject evening primrose cDNA in pBI121(Jefferson et al. 1987 EMLBO J. 6:3901-3907), the followingmanipulations are performed:

[0103] The plasmid pBI121 is digested with EcoICR I (Promega) or Ecl 136II (NEB) and Xba I which excises the GUS coding region and leaves the35S promoter and NOS terminator intact. The evening primroseΔ6-desaturase cDNA is excised from pIB9748-4 by digestion with Xba I andXho I. The Xho I end is made blunt by use of the Klenow fagment. Theexcised gene is then cloned into the Xba I/Eco ICR I sites of pBI121.The resulting construct is used to transform Arabidopsis thaliana viaAgrobacterium according to standard protocols (Bechtold N., Ellis. J.,and Pelletier, G 1993 C. R. Acad Sci Paris 316:1194-1199). Carrot andtobacco are transformed as described above.

1 27 3588 base pairs nucleic acid both linear DNA (genomic) CDS2002..3081 1 GCTAGCCACC AGTGACGATG CCTTGAATTT GGCCATTCTG ACCCAGGCCCGTATTCTGAA 60 TCCCCGCATT CGCATTGTTA ATCGTTTGTT CAACCATGCC CTGGGTAAACGTTTAGACAC 120 CACCTTGCCA GACCACGTTA GTTTGAGTGT TTCCGCCCTG GCGGCCCCGATTTTTTCCTT 180 TGCGGCTTTG GGCAATCAGG CGATCGGGCA ATTGCGTTTG TTTGACCAGACTTGGCCCAT 240 TCAGGAAATT GTCATTCACC AAGACCATCC CTGGCTCAAT TTACCCCTGGCGGATTTATG 300 GGATGATCCG AGCCGAATGT TGATCTATTA CCTACCGGCC CACAGTGAAACGGATTTAGT 360 AGGCGCAGTG GTGAATAATT TAACGTTGCA ATCTGGGGAC CATTTAATAGTGGGACAAAA 420 ACCCCAACCC AAGACCAAAC GGCGATCGCC TTGGCGCAAA TTTTCCAAACTGATTACCAA 480 CCTGCGGGAG TATCAGCGGT ATGTCCAACA GGTGATATGG GTGGTGTTGTTTTTATTGTT 540 GATGATTTTT CTGGCCACCT TCATCTACGT TTCCATTGAT CAACATATTGCCCCAGTGGA 600 CGCGTTGTAT TTTTCCGTGG GCATGATTAC CGGGGCCGGT GGCAAGGAAGAGGTGGCCGA 660 AAAGTCCCCC GATATCATCA AAGTATTCAC AGTGGTGATG ATGATCGCCGGGGCGGGGGT 720 GATTGGTATT TGTTATGCCC TACTGAATGA TTTCATCCTT GGCAGTCGCTTTAGTCAGTT 780 TTTGGATGCG GCCAAGTTAC CCGATCGCCA TCACATCATC ATTTGTGGGCTGGGGGGAGT 840 GAGCATGGCC ATTATTGAAG AGTTAATTCA CCAGGGCCAT GAAATTGTGGTAATCGAAAA 900 GGATACAGAT AATCGTTTCT TGCATACGGC CCGCTCCCTG GGGGTGCCCGTAATTGTGGA 960 GGATGCCCGC CTAGAAAGAA CGTTGGCCTG CGCCAATATC AACCGAGCCGAAGCCATTGT 1020 GGTGGCCACC AGCGACGACA CCGTTAACTT GGAAATTGGC CTAACTGCCAAGGCGATCGC 1080 CCCTAGCCTG CCAGTGGTGT TGCGTTGCCA GGATGCCCAG TTTAGCCTGTCCCTGCAGGA 1140 AGTATTTGAA TTTGAAACGG TGCTTTGTCC GGCGGAATTG GCCACCTATTCCTTTGCGGC 1200 GGCGGCCCTG GGGGGCAAAA TTTTGGGCAA CGGCATGACC GATGATTTGCTGTGGGTAGC 1260 CCTAGCCACC TTAATCACTC CTAACCATCC CTTTGCCGAC CAATTGGTTAAAATTGCAGC 1320 CCAAAAGTCT GATTTCGTTC CCCTCTATCT AGAACGGGGT GGCAAAACCATCCATAGCTG 1380 GGAATTATTG GGTACCCATC TCGACTCTGG AGACGTGTTG TATTTAACCATGCCCGCCAC 1440 TGCCCTAGAG CAACTTTGGC GATCGCCCCG TGCCACTGCT GATCCTCTGGACTCTTTTTT 1500 GGTTTAGCAT GGGGGGATGG AACTCTTGAC TCGGCCCAAT GGTGATCAAGAAAGAACGCT 1560 TTGTCTATGT TTAGTATTTT TAAGTTAACC AACAGCAGAG GATAACTTCCAAAAGAAATT 1620 AAGCTCAAAA AGTAGCAAAA TAAGTTTAAT TCATAACTGA GTTTTACTGCTAAACAGCGG 1680 TGCAAAAAAG TCAGATAAAA TAAAAGCTTC ACTTCGGTTT TATATTGTGACCATGGTTCC 1740 CAGGCATCTG CTCTAGGGAG TTTTTCCGCT GCCTTTAGAG AGTATTTTCTCCAAGTCGGC 1800 TAACTCCCCC ATTTTTAGGC AAAATCATAT ACAGACTATC CCAATATTGCCAGAGCTTTG 1860 ATGACTCACT GTAGAAGGCA GACTAAAATT CTAGCAATGG ACTCCCAGTTGGAATAAATT 1920 TTTAGTCTCC CCCGGCGCTG GAGTTTTTTT GTAGTTAATG GCGGTATAATGTGAAAGTTT 1980 TTTATCTATT TAAATTTATA A ATG CTA ACA GCG GAA AGA ATT AAATTT ACC 2031 Met Leu Thr Ala Glu Arg Ile Lys Phe Thr 1 5 10 CAG AAA CGGGGG TTT CGT CGG GTA CTA AAC CAA CGG GTG GAT GCC TAC 2079 Gln Lys Arg GlyPhe Arg Arg Val Leu Asn Gln Arg Val Asp Ala Tyr 15 20 25 TTT GCC GAG CATGGC CTG ACC CAA AGG GAT AAT CCC TCC ATG TAT CTG 2127 Phe Ala Glu His GlyLeu Thr Gln Arg Asp Asn Pro Ser Met Tyr Leu 30 35 40 AAA ACC CTG ATT ATTGTG CTC TGG TTG TTT TCC GCT TGG GCC TTT GTG 2175 Lys Thr Leu Ile Ile ValLeu Trp Leu Phe Ser Ala Trp Ala Phe Val 45 50 55 CTT TTT GCT CCA GTT ATTTTT CCG GTG CGC CTA CTG GGT TGT ATG GTT 2223 Leu Phe Ala Pro Val Ile PhePro Val Arg Leu Leu Gly Cys Met Val 60 65 70 TTG GCG ATC GCC TTG GCG GCCTTT TCC TTC AAT GTC GGC CAC GAT GCC 2271 Leu Ala Ile Ala Leu Ala Ala PheSer Phe Asn Val Gly His Asp Ala 75 80 85 90 AAC CAC AAT GCC TAT TCC TCCAAT CCC CAC ATC AAC CGG GTT CTG GGC 2319 Asn His Asn Ala Tyr Ser Ser AsnPro His Ile Asn Arg Val Leu Gly 95 100 105 ATG ACC TAC GAT TTT GTC GGGTTA TCT AGT TTT CTT TGG CGC TAT CGC 2367 Met Thr Tyr Asp Phe Val Gly LeuSer Ser Phe Leu Trp Arg Tyr Arg 110 115 120 CAC AAC TAT TTG CAC CAC ACCTAC ACC AAT ATT CTT GGC CAT GAC GTG 2415 His Asn Tyr Leu His His Thr TyrThr Asn Ile Leu Gly His Asp Val 125 130 135 GAA ATC CAT GGA GAT GGC GCAGTA CGT ATG AGT CCT GAA CAA GAA CAT 2463 Glu Ile His Gly Asp Gly Ala ValArg Met Ser Pro Glu Gln Glu His 140 145 150 GTT GGT ATT TAT CGT TTC CAGCAA TTT TAT ATT TGG GGT TTA TAT CTT 2511 Val Gly Ile Tyr Arg Phe Gln GlnPhe Tyr Ile Trp Gly Leu Tyr Leu 155 160 165 170 TTC ATT CCC TTT TAT TGGTTT CTC TAC GAT GTC TAC CTA GTG CTT AAT 2559 Phe Ile Pro Phe Tyr Trp PheLeu Tyr Asp Val Tyr Leu Val Leu Asn 175 180 185 AAA GGC AAA TAT CAC GACCAT AAA ATT CCT CCT TTC CAG CCC CTA GAA 2607 Lys Gly Lys Tyr His Asp HisLys Ile Pro Pro Phe Gln Pro Leu Glu 190 195 200 TTA GCT AGT TTG CTA GGGATT AAG CTA TTA TGG CTC GGC TAC GTT TTC 2655 Leu Ala Ser Leu Leu Gly IleLys Leu Leu Trp Leu Gly Tyr Val Phe 205 210 215 GGC TTA CCT CTG GCT CTGGGC TTT TCC ATT CCT GAA GTA TTA ATT GGT 2703 Gly Leu Pro Leu Ala Leu GlyPhe Ser Ile Pro Glu Val Leu Ile Gly 220 225 230 GCT TCG GTA ACC TAT ATGACC TAT GGC ATC GTG GTT TGC ACC ATC TTT 2751 Ala Ser Val Thr Tyr Met ThrTyr Gly Ile Val Val Cys Thr Ile Phe 235 240 245 250 ATG CTG GCC CAT GTGTTG GAA TCA ACT GAA TTT CTC ACC CCC GAT GGT 2799 Met Leu Ala His Val LeuGlu Ser Thr Glu Phe Leu Thr Pro Asp Gly 255 260 265 GAA TCC GGT GCC ATTGAT GAC GAG TGG GCT ATT TGC CAA ATT CGT ACC 2847 Glu Ser Gly Ala Ile AspAsp Glu Trp Ala Ile Cys Gln Ile Arg Thr 270 275 280 ACG GCC AAT TTT GCCACC AAT AAT CCC TTT TGG AAC TGG TTT TGT GGC 2895 Thr Ala Asn Phe Ala ThrAsn Asn Pro Phe Trp Asn Trp Phe Cys Gly 285 290 295 GGT TTA AAT CAC CAAGTT ACC CAC CAT CTT TTC CCC AAT ATT TGT CAT 2943 Gly Leu Asn His Gln ValThr His His Leu Phe Pro Asn Ile Cys His 300 305 310 ATT CAC TAT CCC CAATTG GAA AAT ATT ATT AAG GAT GTT TGC CAA GAG 2991 Ile His Tyr Pro Gln LeuGlu Asn Ile Ile Lys Asp Val Cys Gln Glu 315 320 325 330 TTT GGT GTG GAATAT AAA GTT TAT CCC ACC TTC AAA GCG GCG ATC GCC 3039 Phe Gly Val Glu TyrLys Val Tyr Pro Thr Phe Lys Ala Ala Ile Ala 335 340 345 TCT AAC TAT CGCTGG CTA GAG GCC ATG GGC AAA GCA TCG TGACATTGCC 3088 Ser Asn Tyr Arg TrpLeu Glu Ala Met Gly Lys Ala Ser 350 355 TTGGGATTGA AGCAAAATGG CAAAATCCCTCGTAAATCTA TGATCGAAGC CTTTCTGTTG 3148 CCCGCCGACC AAATCCCCGA TGCTGACCAAAGGTTGATGT TGGCATTGCT CCAAACCCAC 3208 TTTGAGGGGG TTCATTGGCC GCAGTTTCAAGCTGACCTAG GAGGCAAAGA TTGGGTGATT 3268 TTGCTCAAAT CCGCTGGGAT ATTGAAAGGCTTCACCACCT TTGGTTTCTA CCCTGCTCAA 3328 TGGGAAGGAC AAACCGTCAG AATTGTTTATTCTGGTGACA CCATCACCGA CCCATCCATG 3388 TGGTCTAACC CAGCCCTGGC CAAGGCTTGGACCAAGGCCA TGCAAATTCT CCACGAGGCT 3448 AGGCCAGAAA AATTATATTG GCTCCTGATTTCTTCCGGCT ATCGCACCTA CCGATTTTTG 3508 AGCATTTTTG CCAAGGAATT CTATCCCCACTATCTCCATC CCACTCCCCC GCCTGTACAA 3568 AATTTTATCC ATCAGCTAGC 3588 359amino acids amino acid linear protein 2 Met Leu Thr Ala Glu Arg Ile LysPhe Thr Gln Lys Arg Gly Phe Arg 1 5 10 15 Arg Val Leu Asn Gln Arg ValAsp Ala Tyr Phe Ala Glu His Gly Leu 20 25 30 Thr Gln Arg Asp Asn Pro SerMet Tyr Leu Lys Thr Leu Ile Ile Val 35 40 45 Leu Trp Leu Phe Ser Ala TrpAla Phe Val Leu Phe Ala Pro Val Ile 50 55 60 Phe Pro Val Arg Leu Leu GlyCys Met Val Leu Ala Ile Ala Leu Ala 65 70 75 80 Ala Phe Ser Phe Asn ValGly His Asp Ala Asn His Asn Ala Tyr Ser 85 90 95 Ser Asn Pro His Ile AsnArg Val Leu Gly Met Thr Tyr Asp Phe Val 100 105 110 Gly Leu Ser Ser PheLeu Trp Arg Tyr Arg His Asn Tyr Leu His His 115 120 125 Thr Tyr Thr AsnIle Leu Gly His Asp Val Glu Ile His Gly Asp Gly 130 135 140 Ala Val ArgMet Ser Pro Glu Gln Glu His Val Gly Ile Tyr Arg Phe 145 150 155 160 GlnGln Phe Tyr Ile Trp Gly Leu Tyr Leu Phe Ile Pro Phe Tyr Trp 165 170 175Phe Leu Tyr Asp Val Tyr Leu Val Leu Asn Lys Gly Lys Tyr His Asp 180 185190 His Lys Ile Pro Pro Phe Gln Pro Leu Glu Leu Ala Ser Leu Leu Gly 195200 205 Ile Lys Leu Leu Trp Leu Gly Tyr Val Phe Gly Leu Pro Leu Ala Leu210 215 220 Gly Phe Ser Ile Pro Glu Val Leu Ile Gly Ala Ser Val Thr TyrMet 225 230 235 240 Thr Tyr Gly Ile Val Val Cys Thr Ile Phe Met Leu AlaHis Val Leu 245 250 255 Glu Ser Thr Glu Phe Leu Thr Pro Asp Gly Glu SerGly Ala Ile Asp 260 265 270 Asp Glu Trp Ala Ile Cys Gln Ile Arg Thr ThrAla Asn Phe Ala Thr 275 280 285 Asn Asn Pro Phe Trp Asn Trp Phe Cys GlyGly Leu Asn His Gln Val 290 295 300 Thr His His Leu Phe Pro Asn Ile CysHis Ile His Tyr Pro Gln Leu 305 310 315 320 Glu Asn Ile Ile Lys Asp ValCys Gln Glu Phe Gly Val Glu Tyr Lys 325 330 335 Val Tyr Pro Thr Phe LysAla Ala Ile Ala Ser Asn Tyr Arg Trp Leu 340 345 350 Glu Ala Met Gly LysAla Ser 355 1884 base pairs nucleic acid both linear DNA (genomic) 3AGCTTCACTT CGGTTTTATA TTGTGACCAT GGTTCCCAGG CATCTGCTCT AGGGAGTTTT 60TCCGCTGCCT TTAGAGAGTA TTTTCTCCAA GTCGGCTAAC TCCCCCATTT TTAGGCAAAA 120TCATATACAG ACTATCCCAA TATTGCCAGA GCTTTGATGA CTCACTGTAG AAGGCAGACT 180AAAATTCTAG CAATGGACTC CCAGTTGGAA TAAATTTTTA GTCTCCCCCG GCGCTGGAGT 240TTTTTTGTAG TTAATGGCGG TATAATGTGA AAGTTTTTTA TCTATTTAAA TTTATAAATG 300CTAACAGCGG AAAGAATTAA ATTTACCCAG AAACGGGGGT TTCGTCGGGT ACTAAACCAA 360CGGGTGGATG CCTACTTTGC CGAGCATGGC CTGACCCAAA GGGATAATCC CTCCATGTAT 420CTGAAAACCC TGATTATTGT GCTCTGGTTG TTTTCCGCTT GGGCCTTTGT GCTTTTTGCT 480CCAGTTATTT TTCCGGTGCG CCTACTGGGT TGTATGGTTT TGGCGATCGC CTTGGCGGCC 540TTTTCCTTCA ATGTCGGCCA CGATGCCAAC CACAATGCCT ATTCCTCCAA TCCCCACATC 600AACCGGGTTC TGGGCATGAC CTACGATTTT GTCGGGTTAT CTAGTTTTCT TTGGCGCTAT 660CGCCACAACT ATTTGCACCA CACCTACACC AATATTCTTG GCCATGACGT GGAAATCCAT 720GGAGATGGCG CAGTACGTAT GAGTCCTGAA CAAGAACATG TTGGTATTTA TCGTTTCCAG 780CAATTTTATA TTTGGGGTTT ATATCTTTTC ATTCCCTTTT ATTGGTTTCT CTACGATGTC 840TACCTAGTGC TTAATAAAGG CAAATATCAC GACCATAAAA TTCCTCCTTT CCAGCCCCTA 900GAATTAGCTA GTTTGCTAGG GATTAAGCTA TTATGGCTCG GCTACGTTTT CGGCTTACCT 960CTGGCTCTGG GCTTTTCCAT TCCTGAAGTA TTAATTGGTG CTTCGGTAAC CTATATGACC 1020TATGGCATCG TGGTTTGCAC CATCTTTATG CTGGCCCATG TGTTGGAATC AACTGAATTT 1080CTCACCCCCG ATGGTGAATC CGGTGCCATT GATGACGAGT GGGCTATTTG CCAAATTCGT 1140ACCACGGCCA ATTTTGCCAC CAATAATCCC TTTTGGAACT GGTTTTGTGG CGGTTTAAAT 1200CACCAAGTTA CCCACCATCT TTTCCCCAAT ATTTGTCATA TTCACTATCC CCAATTGGAA 1260AATATTATTA AGGATGTTTG CCAAGAGTTT GGTGTGGAAT ATAAAGTTTA TCCCACCTTC 1320AAAGCGGCGA TCGCCTCTAA CTATCGCTGG CTAGAGGCCA TGGGCAAAGC ATCGTGACAT 1380TGCCTTGGGA TTGAAGCAAA ATGGCAAAAT CCCTCGTAAA TCTATGATCG AAGCCTTTCT 1440GTTGCCCGCC GACCAAATCC CCGATGCTGA CCAAAGGTTG ATGTTGGCAT TGCTCCAAAC 1500CCACTTTGAG GGGGTTCATT GGCCGCAGTT TCAAGCTGAC CTAGGAGGCA AAGATTGGGT 1560GATTTTGCTC AAATCCGCTG GGATATTGAA AGGCTTCACC ACCTTTGGTT TCTACCCTGC 1620TCAATGGGAA GGACAAACCG TCAGAATTGT TTATTCTGGT GACACCATCA CCGACCCATC 1680CATGTGGTCT AACCCAGCCC TGGCCAAGGC TTGGACCAAG GCCATGCAAA TTCTCCACGA 1740GGCTAGGCCA GAAAAATTAT ATTGGCTCCT GATTTCTTCC GGCTATCGCA CCTACCGATT 1800TTTGAGCATT TTTGCCAAGG AATTCTATCC CCACTATCTC CATCCCACTC CCCCGCCTGT 1860ACAAAATTTT ATCCATCAGC TAGC 1884 1685 base pairs nucleic acid both linearDNA (genomic) 4 AATATCTGCC TACCCTCCCA AAGAGAGTAG TCATTTTTCA TCAATGGCTGCTCAAATCAA 60 GAAATACATT ACCTCAGATG AACTCAAGAA CCACGATAAA CCCGGAGATCTATGGATCTC 120 GATTCAAGGG AAAGCCTATG ATGTTTCGGA TTGGGTGAAA GACCATCCAGGTGGCAGCTT 180 TCCCTTGAAG AGTCTTGCTG GTCAAGAGGT AACTGATGCA TTTGTTGCATTCCATCCTGC 240 CTCTACATGG AAGAATCTTG ATAAGTTTTT CACTGGGTAT TATCTTAAAGATTACTCTGT 300 TTCTGAGGTT TCTAAAGATT ATAGGAAGCT TGTGTTTGAG TTTTCTAAAATGGGTTTGTA 360 TGACAAAAAA GGTCATATTA TGTTTGCAAC TTTGTGCTTT ATAGCAATGCTGTTTGCTAT 420 GAGTGTTTAT GGGGTTTTGT TTTGTGAGGG TGTTTTGGTA CATTTGTTTTCTGGGTGTTT 480 GATGGGGTTT CTTTGGATTC AGAGTGGTTG GATTGGACAT GATGCTGGGCATTATATGGT 540 AGTGTCTGAT TCAAGGCTTA ATAAGTTTAT GGGTATTTTT GCTGCAAATTGTCTTTCAGG 600 AATAAGTATT GGTTGGTGGA AATGGAACCA TAATGCACAT CACATTGCCTGTAATAGCCT 660 TGAATATGAC CCTGATTTAC AATATATACC ATTCCTTGTT GTGTCTTCCAAGTTTTTTGG 720 TTCACTCACC TCTCATTTCT ATGAGAAAAG GTTGACTTTT GACTCTTTATCAAGATTCTT 780 TGTAAGTTAT CAACATTGGA CATTTTACCC TATTATGTGT GCTGCTAGGCTCAATATGTA 840 TGTACAATCT CTCATAATGT TGTTGACCAA GAGAAATGTG TCCTATCGAGCTCAGGAACT 900 CTTGGGATGC CTAGTGTTCT CGATTTGGTA CCCGTTGCTT GTTTCTTGTTTGCCTAATTG 960 GGGTGAAAGA ATTATGTTTG TTATTGCAAG TTTATCAGTG ACTGGAATGCAACAAGTTCA 1020 GTTCTCCTTG AACCACTTCT CTTCAAGTGT TTATGTTGGA AAGCCTAAAGGGAATAATTG 1080 GTTTGAGAAA CAAACGGATG GGACACTTGA CATTTCTTGT CCTCCTTGGATGGATTGGTT 1140 TCATGGTGGA TTGCAATTCC AAATTGAGCA TCATTTGTTT CCCAAGATGCCTAGATGCAA 1200 CCTTAGGAAA ATCTCGCCCT ACGTGATCGA GTTATGCAAG AAACATAATTTGCCTTACAA 1260 TTATGCATCT TTCTCCAAGG CCAATGAAAT GACACTCAGA ACATTGAGGAACACAGCATT 1320 GCAGGCTAGG GATATAACCA AGCCGCTCCC GAAGAATTTG GTATGGGAAGCTCTTCACAC 1380 TCATGGTTAA AATTACCCTT AGTTCATGTA ATAATTTGAG ATTATGTATCTCCTATGTTT 1440 GTGTCTTGTC TTGGTTCTAC TTGTTGGAGT CATTGCAACT TGTCTTTTATGGTTTATTAG 1500 ATGTTTTTTA ATATATTTTA GAGGTTTTGC TTTCATCTCC ATTATTGATGAATAAGGAGT 1560 TGCATATTGT CAATTGTTGT GCTCAATATC TGATATTTTG GAATGTACTTTGTACCACTG 1620 TGTTTTCAGT TGAAGCTCAT GTGTACTTCT ATAGACTTTG TTTAAATGGTTATGTCATGT 1680 TATTT 1685 448 amino acids amino acid linear Protein 5Met Ala Ala Gln Ile Lys Lys Tyr Ile Thr Ser Asp Glu Leu Lys Asn 1 5 1015 His Asp Lys Pro Gly Asp Leu Trp Ile Ser Ile Gln Gly Lys Ala Tyr 20 2530 Asp Val Ser Asp Trp Val Lys Asp His Pro Gly Gly Ser Phe Pro Leu 35 4045 Lys Ser Leu Ala Gly Gln Glu Val Thr Asp Ala Phe Val Ala Phe His 50 5560 Pro Ala Ser Thr Trp Lys Asn Leu Asp Lys Phe Phe Thr Gly Tyr Tyr 65 7075 80 Leu Lys Asp Tyr Ser Val Ser Glu Val Ser Lys Asp Tyr Arg Lys Leu 8590 95 Val Phe Glu Phe Ser Lys Met Gly Leu Tyr Asp Lys Lys Gly His Ile100 105 110 Met Phe Ala Thr Leu Cys Phe Ile Ala Met Leu Phe Ala Met SerVal 115 120 125 Tyr Gly Val Leu Phe Cys Glu Gly Val Leu Val His Leu PheSer Gly 130 135 140 Cys Leu Met Gly Phe Leu Trp Ile Gln Ser Gly Trp IleGly His Asp 145 150 155 160 Ala Gly His Tyr Met Val Val Ser Asp Ser ArgLeu Asn Lys Phe Met 165 170 175 Gly Ile Phe Ala Ala Asn Cys Leu Ser GlyIle Ser Ile Gly Trp Trp 180 185 190 Lys Trp Asn His Asn Ala His His IleAla Cys Asn Ser Leu Glu Tyr 195 200 205 Asp Pro Asp Leu Gln Tyr Ile ProPhe Leu Val Val Ser Ser Lys Phe 210 215 220 Phe Gly Ser Leu Thr Ser HisPhe Tyr Glu Lys Arg Leu Thr Phe Asp 225 230 235 240 Ser Leu Ser Arg PhePhe Val Ser Tyr Gln His Trp Thr Phe Tyr Pro 245 250 255 Ile Met Cys AlaAla Arg Leu Asn Met Tyr Val Gln Ser Leu Ile Met 260 265 270 Leu Leu ThrLys Arg Asn Val Ser Tyr Arg Ala Gln Glu Leu Leu Gly 275 280 285 Cys LeuVal Phe Ser Ile Trp Tyr Pro Leu Leu Val Ser Cys Leu Pro 290 295 300 AsnTrp Gly Glu Arg Ile Met Phe Val Ile Ala Ser Leu Ser Val Thr 305 310 315320 Gly Met Gln Gln Val Gln Phe Ser Leu Asn His Phe Ser Ser Ser Val 325330 335 Tyr Val Gly Lys Pro Lys Gly Asn Asn Trp Phe Glu Lys Gln Thr Asp340 345 350 Gly Thr Leu Asp Ile Ser Cys Pro Pro Trp Met Asp Trp Phe HisGly 355 360 365 Gly Ser Gln Phe Gln Ile Glu His His Leu Phe Pro Lys MetPro Arg 370 375 380 Cys Asn Leu Arg Lys Ile Ser Pro Tyr Val Ile Glu LeuCys Lys Lys 385 390 395 400 His Asn Leu Pro Tyr Asn Tyr Ala Ser Phe SerLys Ala Asn Glu Met 405 410 415 Thr Leu Arg Thr Leu Arg Asn Thr Ala LeuGln Ala Arg Asp Ile Thr 420 425 430 Lys Pro Leu Pro Lys Asn Leu Val TrpGlu Ala Leu His Thr His Gly 435 440 445 8 amino acids amino acid linearPeptide 6 Trp Ile Gly His Asp Ala Gly His 1 5 8 amino acids amino acidlinear Peptide 7 Asn Val Gly His Asp Ala Asn His 1 5 8 amino acids aminoacid linear Peptide 8 Val Leu Gly His Asp Cys Gly His 1 5 8 amino acidsamino acid linear DNA (peptide) 9 Val Ile Ala His Glu Cys Gly His 1 5 8amino acids amino acid linear Peptide 10 Val Ile Gly His Asp Cys Ala His1 5 8 amino acids amino acid linear Peptide 11 Val Val Gly His Asp CysGly His 1 5 5 amino acids amino acid linear Peptide 12 His Asn Ala HisHis 1 5 6 amino acids amino acid linear DNA (peptide) 13 His Asn Tyr LeuHis His 1 5 5 amino acids amino acid linear Peptide 14 His Arg Thr HisHis 1 5 5 amino acids amino acid linear Peptide 15 His Arg Arg His His 15 5 amino acids amino acid linear Peptide 16 His Asp Arg His His 1 5 5amino acids amino acid linear Peptide 17 His Asp Gln His His 1 5 5 aminoacids amino acid linear Peptide 18 His Asp His His His 1 5 5 amino acidsamino acid linear Peptide 19 His Asn His His His 1 5 6 amino acids aminoacid linear Peptide 20 Phe Gln Ile Glu His His 1 5 6 amino acids aminoacid linear Peptide 21 His Gln Val Thr His His 1 5 5 amino acids aminoacid linear Peptide 22 His Val Ile His His 1 5 5 amino acids amino acidlinear Peptide 23 His Val Ala His His 1 5 5 amino acids amino acidlinear Peptide 24 His Ile Pro His His 1 5 5 amino acids amino acidlinear Peptide 25 His Val Pro His His 1 5 1702 base pairs nucleic acidboth linear DNA (genomic) CDS 48..1406 CDS 48..1406 26 CCCCAAAAATTTTCATTGTT CTCCATCTGG ACCACAGCAT CCACACA ATG GAG GGC 56 Met Glu Gly 1GAA GCT AAG AAG TAT ATC ACG GCG GAG GAC CTC CGC CGC CAC AAC AAG 104 GluAla Lys Lys Tyr Ile Thr Ala Glu Asp Leu Arg Arg His Asn Lys 5 10 15 TCCGGC GAT CTC TGG ATC TCC ATC CAG GGC AAG GTC TAC GAC TGC TCT 152 Ser GlyAsp Leu Trp Ile Ser Ile Gln Gly Lys Val Tyr Asp Cys Ser 20 25 30 35 CGGTGG GCG GCG GAG CAC CCC GGC GGC GAG GTC CCG CTC CTC AGT CTG 200 Arg TrpAla Ala Glu His Pro Gly Gly Glu Val Pro Leu Leu Ser Leu 40 45 50 GCC GGCCAG GAC GTC ACC GAC GCC TTC ATT GCG TAC CAC CCG GGC ACG 248 Ala Gly GlnAsp Val Thr Asp Ala Phe Ile Ala Tyr His Pro Gly Thr 55 60 65 GCG TGG CGGCAT CTG GAT CCG CTC TTC ACC GGC TAC TAC TAC CTC AAG 296 Ala Trp Arg HisLeu Asp Pro Leu Phe Thr Gly Tyr Tyr Tyr Leu Lys 70 75 80 GAC TTC GAA GTGTCG GAG ATC TCC AAG GAC TAC CGG AGG CTT TTG AAC 344 Asp Phe Glu Val SerGlu Ile Ser Lys Asp Tyr Arg Arg Leu Leu Asn 85 90 95 GAG ATG TCG CGG TCCGGG ATC TTC GAG AAG AAG GGC CAC CAC ATC ATG 392 Glu Met Ser Arg Ser GlyIle Phe Glu Lys Lys Gly His His Ile Met 100 105 110 115 TGG ACG TTC GTCGGC GTT GCG GTC ATG ATG GCG GCA ATC GTC TAC GGC 440 Trp Thr Phe Val GlyVal Ala Val Met Met Ala Ala Ile Val Tyr Gly 120 125 130 GTG CTG GCG TCGGAG TCC GTC GGA GTT CAC ATG CTC TGC GGC GCA CTG 488 Val Leu Ala Ser GluSer Val Gly Val His Met Leu Cys Gly Ala Leu 135 140 145 CTG GGC TTG CTGTGG ATC CAA GCC GCG TAT GTG GGC CAT GAC TCC GGC 536 Leu Gly Leu Leu TrpIle Gln Ala Ala Tyr Val Gly His Asp Ser Gly 150 155 160 CAT TAC CAG GTGATG CCA ACC CGT GGA TAC AAC AGA ATC ACG CAA CTC 584 His Tyr Gln Val MetPro Thr Arg Gly Tyr Asn Arg Ile Thr Gln Leu 165 170 175 ATA GCA GGC AACATC CTA ACC GGA ATC AGC ATC GCG TGG TGG AAG TGG 632 Ile Ala Gly Asn IleLeu Thr Gly Ile Ser Ile Ala Trp Trp Lys Trp 180 185 190 195 ACC CAC AACGCC CAC CAC CTC GCC TGC AAC AGC CTC GAC TAC GAC CCC 680 Thr His Asn AlaHis His Leu Ala Cys Asn Ser Leu Asp Tyr Asp Pro 200 205 210 GAC CTC CAGCAC ATC CCC GTA TTC GCC GTC TCC ACC CGA CTC TTC AAC 728 Asp Leu Gln HisIle Pro Val Phe Ala Val Ser Thr Arg Leu Phe Asn 215 220 225 TCC ATC ACCTCG GTC TTC TAT GGC CGA GTC CTG AAA TTC GAC GAA GTG 776 Ser Ile Thr SerVal Phe Tyr Gly Arg Val Leu Lys Phe Asp Glu Val 230 235 240 GCA CGG TTCCTA GTC AGC TAC CAG CAC TGG ACC TAC TAC CCG GTC ATG 824 Ala Arg Phe LeuVal Ser Tyr Gln His Trp Thr Tyr Tyr Pro Val Met 245 250 255 ATC TTC GGCCGA GTC AAC CTC TTC ATC CAG ACC TTT TTA TTG CTC CTC 872 Ile Phe Gly ArgVal Asn Leu Phe Ile Gln Thr Phe Leu Leu Leu Leu 260 265 270 275 ACC AGGCGC GAC GTC CCT GAC CGC GCT CTA AAC TTA ATG GGT ATC GCG 920 Thr Arg ArgAsp Val Pro Asp Arg Ala Leu Asn Leu Met Gly Ile Ala 280 285 290 GTT TTCTGG ACG TGG TTC CCG CTC TTC GTA TCT TGT CTC CCG AAC TGG 968 Val Phe TrpThr Trp Phe Pro Leu Phe Val Ser Cys Leu Pro Asn Trp 295 300 305 CCT GAACGG TTC GGG TTC GTC CTC ATC AGC TTT GCG GTC ACG GCG ATC 1016 Pro Glu ArgPhe Gly Phe Val Leu Ile Ser Phe Ala Val Thr Ala Ile 310 315 320 CAG CACGTC CAG TTC ACG CTC AAC CAC TTC TCC GGC GAC ACA TAC GTG 1064 Gln His ValGln Phe Thr Leu Asn His Phe Ser Gly Asp Thr Tyr Val 325 330 335 GGC CCCCCC AAG GGC GAC AAC TGG TTC GAG AAG CAG ACG AAA GGG ACG 1112 Gly Pro ProLys Gly Asp Asn Trp Phe Glu Lys Gln Thr Lys Gly Thr 340 345 350 355 ATCGAT ATC ACG TGC CCA CCG TGG ATG GAC TGG TTC TTT GGT GGG CTG 1160 Ile AspIle Thr Cys Pro Pro Trp Met Asp Trp Phe Phe Gly Gly Leu 360 365 370 CAGTTC CAG TTG GAG CAC CAC TTG TTC CCT AGG CTG CCG CGT GGG CAG 1208 Gln PheGln Leu Glu His His Leu Phe Pro Arg Leu Pro Arg Gly Gln 375 380 385 CTTAGG AAG ATT GCG CCC TTG GCT CGG GAC TTG TGT AAG AAG CAC GGG 1256 Leu ArgLys Ile Ala Pro Leu Ala Arg Asp Leu Cys Lys Lys His Gly 390 395 400 ATGCCG TAT AGG AGC TTC GGG TTT TGG GAC GAC GCT AAT GTC AGG ACA 1304 Met ProTyr Arg Ser Phe Gly Phe Trp Asp Asp Ala Asn Val Arg Thr 405 410 415 ATTCGG ACG CTG AGG GAT GCG GCG GTT CAG GCG CGT GAC CTT AAT TCG 1352 Ile ArgThr Leu Arg Asp Ala Ala Val Gln Ala Arg Asp Leu Asn Ser 420 425 430 435GCC CCG TGC CCT AAG AAA CTT GGG TAT GGG GAA GCT TAT AAC ACC CAT 1400 AlaPro Cys Pro Lys Lys Leu Gly Tyr Gly Glu Ala Tyr Asn Thr His 440 445 450GGT TGA TTGTGGTTTT GTGTTGTGGG TTGGAGGATC TTCTTATTAT TGATTTATGT 1456Gly * CCACAATATT GAACTGAATA ACCATGGAAG GCACTACGTT CAGCTTAACT TTGCTTAACT1516 TTGCTAGCTG GTTGCGTTCC CTTGTTGGGG GCAAAGTGCA GTATTTATTT TCTTATCCCA1576 TGTACTTTTT GATTATTGTT CTTATTCGTA TCATAAATAA TTTATTATTG ATTAATTTTT1636 GTTGTAGTTG GGTGTCTATA GCAAGTTTAT AATACTGAGA TATATTTTTT TGGTAAAAAA1696 AAAAAA 1702 452 amino acids amino acid linear protein 27 Met GluGly Glu Ala Lys Lys Tyr Ile Thr Ala Glu Asp Leu Arg Arg 1 5 10 15 HisAsn Lys Ser Gly Asp Leu Trp Ile Ser Ile Gln Gly Lys Val Tyr 20 25 30 AspCys Ser Arg Trp Ala Ala Glu His Pro Gly Gly Glu Val Pro Leu 35 40 45 LeuSer Leu Ala Gly Gln Asp Val Thr Asp Ala Phe Ile Ala Tyr His 50 55 60 ProGly Thr Ala Trp Arg His Leu Asp Pro Leu Phe Thr Gly Tyr Tyr 65 70 75 80Tyr Leu Lys Asp Phe Glu Val Ser Glu Ile Ser Lys Asp Tyr Arg Arg 85 90 95Leu Leu Asn Glu Met Ser Arg Ser Gly Ile Phe Glu Lys Lys Gly His 100 105110 His Ile Met Trp Thr Phe Val Gly Val Ala Val Met Met Ala Ala Ile 115120 125 Val Tyr Gly Val Leu Ala Ser Glu Ser Val Gly Val His Met Leu Cys130 135 140 Gly Ala Leu Leu Gly Leu Leu Trp Ile Gln Ala Ala Tyr Val GlyHis 145 150 155 160 Asp Ser Gly His Tyr Gln Val Met Pro Thr Arg Gly TyrAsn Arg Ile 165 170 175 Thr Gln Leu Ile Ala Gly Asn Ile Leu Thr Gly IleSer Ile Ala Trp 180 185 190 Trp Lys Trp Thr His Asn Ala His His Leu AlaCys Asn Ser Leu Asp 195 200 205 Tyr Asp Pro Asp Leu Gln His Ile Pro ValPhe Ala Val Ser Thr Arg 210 215 220 Leu Phe Asn Ser Ile Thr Ser Val PheTyr Gly Arg Val Leu Lys Phe 225 230 235 240 Asp Glu Val Ala Arg Phe LeuVal Ser Tyr Gln His Trp Thr Tyr Tyr 245 250 255 Pro Val Met Ile Phe GlyArg Val Asn Leu Phe Ile Gln Thr Phe Leu 260 265 270 Leu Leu Leu Thr ArgArg Asp Val Pro Asp Arg Ala Leu Asn Leu Met 275 280 285 Gly Ile Ala ValPhe Trp Thr Trp Phe Pro Leu Phe Val Ser Cys Leu 290 295 300 Pro Asn TrpPro Glu Arg Phe Gly Phe Val Leu Ile Ser Phe Ala Val 305 310 315 320 ThrAla Ile Gln His Val Gln Phe Thr Leu Asn His Phe Ser Gly Asp 325 330 335Thr Tyr Val Gly Pro Pro Lys Gly Asp Asn Trp Phe Glu Lys Gln Thr 340 345350 Lys Gly Thr Ile Asp Ile Thr Cys Pro Pro Trp Met Asp Trp Phe Phe 355360 365 Gly Gly Leu Gln Phe Gln Leu Glu His His Leu Phe Pro Arg Leu Pro370 375 380 Arg Gly Gln Leu Arg Lys Ile Ala Pro Leu Ala Arg Asp Leu CysLys 385 390 395 400 Lys His Gly Met Pro Tyr Arg Ser Phe Gly Phe Trp AspAsp Ala Asn 405 410 415 Val Arg Thr Ile Arg Thr Leu Arg Asp Ala Ala ValGln Ala Arg Asp 420 425 430 Leu Asn Ser Ala Pro Cys Pro Lys Lys Leu GlyTyr Gly Glu Ala Tyr 435 440 445 Asn Thr His Gly 450

What is claimed:
 1. An isolated nucleic acid encoding an eveningprimrose Δ6-desaturase.
 2. The isolated nucleic acid of claim 1comprising at least one of the nucleotide sequence of SEQ ID NO: 26 ornucleotides 49 to 1401 of SEQ ID NO:
 26. 3. An isolated nucleic acidthat codes for the amino acid sequence of SEQ ID NO:
 27. 4. A vectorcomprising the nucleic acid of any one claims 1-3.
 5. An expressionvector comprising the isolated nucleic acid of any one of claims 1-3operably linked to a promoter which effects expression of the geneproduct of said isolated nucleic acid.
 6. An expression vectorcomprising the isolated nucleic acid of any one of claims 1-3 operablylinked to a promoter and a termination signal capable of effectingexpression of the gene product of said isolated nucleic acid.
 7. Theexpression vector of claim 5 wherein said promoter is a Δ6-desaturasepromoter, an Anabaena carboxylase promoter, a helianthinin promoter, aglycinin in promoter, a napin promoter, the 35S promoter from CaMV, ahelianthinin tissue-specific promoter, an oleosin seed-specificpromoter, or an albumin seed-specific promoter.
 8. The expression vectorof claim 6 wherein said promoter is a Δ6-desaturase promoter, anAnabaena carboxylase promoter, a helianthinin promoter, a glycininpromoter, a napin promoter, the 35S promoter from CaMV, a helianthinintissue-specific promoter, an oleosin seed-specific promoter, or analbumin seed-specific promoter.
 9. An expression vector comprising theisolated nucleic acid of any one of claims 1-3 operably linked to aconsitutive promoter.
 10. An expression vector comprising the isolatednucleic acid of any one of claims 1-3 operably linked to a tissuespecific promoter.
 11. The expression vector of claim 6 wherein saidtermination signal is a Synechocystis termination signal, a nopalinesynthase termination signal, or a seed termination signal.
 12. A cellcomprising the vector of claim
 4. 13. A cell comprising the vector ofclaim
 5. 14. A cell comprising the vector of claim
 6. 15. The cell ofclaim 12 wherein said cell is an animal cell, a bacterial cell, a plantcell or a fungal cell.
 16. The cell of claim 13 wherein said cell is ananimal cell, a bacterial cell, a plant cell or a fungal cell.
 17. Thecell of claim 14 wherein said cell is an animal cell, a bacterial cell,a plant cell or a fungal cell.
 18. A transgenic bacterium or plantcomprising the isolated nucleic acid of any one of claims 1-3.
 19. Atransgenic bacterium or plant comprising the vector of claim
 4. 20. Atransgenic bacterium or plant comprising the vector of claim
 5. 21. Atransgenic bacterium or plant comprising the vector of claim
 6. 22. Aplant or progeny of said plant which has been regenerated from the plantcell of claim
 15. 23. The plant of claim 22 wherein said plant is asunflower, soybean, maize, tobacco, peanut, carrot or oil seed rapeplant.
 24. A method of producing a plant with increased gamma linolenicacid (GLA) content which comprises: (a) transforming a plant cell withthe isolated nucleic acid of any one of claims 1-3; and (b) regeneratinga plant with increased GLA content from said plant cell.
 25. A method ofproducing a plant with increased gamma linolenic acid (GLA) contentwhich comprises: (a) transforming a plant cell with the vector of claim4; and (b) regenerating a plant with increased GLA content from saidplant cell.
 26. A method of producing a plant with increased gammalinolenic acid (GLA) content which comprises: (a) transforming a plantcell with the vector of claim 5; and (b) regenerating a plant withincreased GLA content from said plant cell.
 27. A method of producing aplant with increased gamma linolenic acid (GLA) content which comprises:(a) transforming a plant cell with the vector of claim 6; and (b)regenerating a plant with increased GLA content from said plant cell.28. The method of claim 24 wherein said plant is a sunflower, soybean,maize, tobacco, peanut, carrot or oil seed rape plant.
 29. The method ofclaim 25 wherein said plant is a sunflower, soybean, maize, tobacco,peanut, carrot or oil seed rape plant.
 30. The method of claim 26wherein said plant is a sunflower, soybean, maize, tobacco, peanut,carrot or oil seed rape plant.
 31. The method of claim 27 wherein saidplant is a sunflower, soybean, maize, tobacco, peanut, carrot or oilseed rape plant.
 32. A method of inducing or increasing production ofgamma linolenic acid (GLA) in an organism lacking in or producing lowlevels of GLA which comprises transforming said organism with theisolated nucleic acid of any one of claims 1-3.
 33. A method of inducingor increasing production of gamma linolenic acid (GLA) in an organismdeficient or lacking in or producing low levels of GLA which comprisestransforming said organism with the vector of claim
 4. 34. A method ofinducing or increasing production of gamma linolenic acid (GLA) in anorganism deficient or lacking in or producing low levels of GLA whichcomprises transforming said organism with the vector of claim
 5. 35. Amethod of inducing or increasing production of gamma linolenic acid(GLA) in an organism deficient or lacking in or producing low levels ofGLA which comprises transforming said organism with the vector of claim6.
 36. A method of inducing production of gamma linolenic acid (GLA) inan organism deficient or lacking in or producing low levels of GLA andlinoleic acid (LA) which comprises transforming said organism with anisolated nucleic acid encoding bacterial Δ6-desaturase and an isolatednucleic acid encoding Δ12-desaturase.
 37. A method of inducingproduction of gamma linolenic acid (GLA) in an organism deficient orlacking in or producing low levels of GLA and linoleic acid (LA) whichcomprises transforming said organism with at least one expression vectorcomprising an isolated nucleic acid encoding evening primroseΔ6-desaturase and an isolated nucleic acid encoding Δ12desaturase. 38.The method of inducing production of octadecatetraeonic acid in at leastone of a plant deficient or lacking in or producing low levels ofoctadecatetraenoic acid, a bacterium which produces α-linolenic acid, ora bacterium which exhibits a Δ15-desaturase activity on a GLA substratewhich comprises transforming said plant or bacterium with any one ofclaims 1-3.
 39. A method of inducing production of octadecatetraeonicacid in at least one of a plant deficient or lacking in or producing lowlevels of octadecatetraenoic acid, a bacterium which producesα-linolenic acid, or a bacterium which exhibits a Δ15-desaturaseactivity on a GLA substrate which comprises transforming said plant orbacterium with the vector of claim
 4. 40. A method of inducingproduction of octadecatetraeonic acid in at least one of a plantdeficient or lacking in or producing low levels of octadecatetraenoicacid, a bacterium which produces α-linolenic acid, or a bacterium whichexhibits a Δ15-desaturase activity on a GLA substrate which comprisestransforming said plant or bacterium with the vector of claim
 5. 41. Amethod of inducing production of octadecatetraeonic acid in at least oneof a plant deficient or lacking in or producing low levels ofoctadecatetraenoic acid, a bacterium which produces α-linolenic acid, ora bacterium which exhibits a Δ15-desaturase activity on a GLA substratewhich comprises transforming said plant or bacterium with the vector ofclaim
 6. 42. A method of inducing production of octadecatetraeonic acidin at least one of a plant deficient or lacking in or producing lowlevels of octadecatetraenoic acid, a bacterium which producesα-linolenic acid, or a bacterium which exhibits a Δ15-desaturaseactivity on a GLA substrate which comprises transforming said plant orbacterium with the vector of claim
 7. 43. The method of claim 40 whereinsaid plant is a sunflower, soybean, maize, tobacco, peanut, carrot oroil seed rape plant.
 44. The method of claim 41 wherein said plant is asunflower, soybean, maize, tobacco, peanut, carrot or oil seed rapeplant.
 45. The method of claim 42 wherein said plant is a sunflower,soybean, maize, tobacco, peanut, carrot or oil seed rape plant.
 46. Themethod of claim 43 wherein said plant is a sunflower, soybean, maize,tobacco, peanut, carrot or oil seed rape plant.